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Chemistry 422

BIOCHEMISTRY

LABORATORYMANUAL

Mark Brandt , Ph.D.

Thir d edit ionJ a nuar y , 2002

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Table of Contents

Int roduction................................................................................................. 4

Keeping a La bora tory Notebook.... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 6

La bora tory Reports................................................................................... 8

Experiment 1: Introduction to Techniques. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 17

U se of pipetmen................................................................................. 17

Spectroscopy a nd dilutions.............................................................. 20

Ana lysis of experiment 1 results.................................................... 25

Experiment 2: Protein Purification................................................. 26

P urifica tion of LD H........................................................................... 33

P urifica tion of LD H (continued)..................................................... 37

LDH Enzym e assa ys... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 41P rotein a ssa ys................................................................................... 44

Ca lculation hints: P urificat ion t able.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 46

Experiment 3: Characterization of LDH ........................................ 48

SD S P AG E.......................................................................................... 48

Western blott ing................................................................................ 57

Western blott ing (cont inued)........................................................... 62

G el filtra tion chromatography... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . 66

G el filtra tion chromatography (continued)... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 71

P rotein crysta llogra phy................................................................... 73

Experiment 4: Enzyme Kinetics........................................................ 77

Kmdetermina tion.............................................................................. 87

Lac t a t e Kmdeterminat ion (continued)... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 90

P yruva t e Kmdeterminat ion........................................................... 92

In hibition kinetics.............................................................................. 94

Inh ibitor type determina tion........................................................... 100

Ch emica l modifica tion of LD H........................................................ 102

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Table of Contents (continued)

Experiment 5:Cloning of LDH........................................................... 104

P CR a nd pla smid prepara tion........................................................ 107Aga rose gels a nd restr iction digests.............................................. 114

Liga tion a nd tra nsformat ion........................................................... 118

Selection and screening.................................................................... 122

Screening a nd sequencing................................................................ 125

Activity measu rements................................................................... 130

Definitions................................................................................................. 131

Other useful Information:B iochemistr y St ockroom: MH -277Ch emistr y & B iochemistry Office: MH -580

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Introduction to the Laboratory

This course is int ended t o int roduce you to some of th e most w idely used experiment a lprocedures in biochemistry, including protein purification and characterization,enzyme assa ys a nd kinetics, and DNA isolat ion a nd ma nipula tion. You w ill a lso gainsome familiarity with some of the types of equipment frequently used inbiochemistry.

Resea rch is often a collaborat ive effort in w hich m a ny people ma y cont ribute todiff eren t a spects of a given project. F ew pa pers in th e biochemica l litera ture a rewrit t en by single authors; the vast ma jority of pa pers ha ve at least tw o authors, andma ny papers ha ve more th a n t en contributing people. In pa rt to provide a morea uth entic experience of a ctua l lab w ork, experiment s w ill be done in groups of tw o orth ree. You ma y choose pa rt ners, or you can a sk to be assigned t o a group.

P rior t o each lab period, you will need to spend some t ime reading t he La bora toryManual. This reading will provide background information and an outline of the

procedures to be performed. If y ou do not do this, you w ill find y ourself w a st ing la rgea mounts of class t ime, a nd a nnoying both y our lab pa rtn ers and your instructor. Youwill a lso f ind i t dif ficult to a nsw er the prelab q uest ions t ha t m ust be turn ed in eachday .

The biochemist ry la borat ory course, like all la borat ory courses, is a n explora tion ofprocedures. This m ean s th a t, in order t o get full benefit from t he course, you w ill needto read the manual, and you should participate as much as possible in thediscussions. You should ask questions in or out of class. You should also try topart icipat e in the actua l la b work (a nd not simply allow y our lab par tn ers to do th ingsfor you). The more effort y ou put int o the cours e work, t he more you w ill lear n. Theclass is a n opportun ity t o lea rn va luable skills; take full a dva nta ge of it!

SAFETY: La bora tories conta in ha za rds of va rious kinds. Everyoneis r equ i r edt owear closed-toe shoes, long pants, goggles with side shields, and a lab coatw hile performing laborat ory w ork. Stud ents should not w ork in the labora tory if theinstr uctor is not present.

Some of th e chemicals used a re toxic, mut a genic, or t era togenic. If you believe th a tyou ha ve a h ealt h condit ion t ha t put s you at exceptiona l risk, or believe yourself to bepregna nt, plea se see your instructor in privat e to discuss t he issue. If you ha vequest ions or concerns a bout exposure to ha za rdous chemica ls , plea se consult yourinst ructor or go to th e Resea rch a nd In str uctiona l Sa fety Office (MH-557).

PHILOSOPHICAL ISSUES: Scientific research involves an exploration of theunknown . In some classes, a q uestion ha s a sin gle correct a nsw er, which is know nto th e instr uctor , and impa rt ed to th e stud ents. In research, however, the correctanswer is rarely known ahead of t ime, and must instead be inferred from theexperimenta l results . Resear chers must th erefore become accust omed t o some levelof uncer ta inty a bout t he correct a nsw er to an y exper imenta l quest ion, a nd m usta lwa ys rema in open to experimenta l evidence th a t cont ra dicts a hypothesis th a t ha s

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a risen from previous experiment s. Your t a sk a s a scientist w ill be to consider yourda ta , and to a t tempt to interpret i t . In this context , wrong a nsw ers a re answ erstha t a re contra dicted by your da ta or tha t do not a r ise log ica l ly from the da t a youhave collected.

This uncer ta inty a s to the correct a nsw er means t ha t you must be ca re fu l w hen

report ing w ha t you did an d wh a t you observed, especia lly if you observe somethingunexpected. Humans are good at fooling themselves; you need to guard againstreport ing w ha t you expect to seer a t her tha n wha t you ac tua l ly d i dsee. Scientificfraud, in which people intentionally report false data, is considered very seriousbeca use it results in a difficult-to-overcome belief in an a nsw er th a t conflicts w ith th etruth. You will occasionally see retractions, in which a scientist publishes astatement that information in a previously published paper is the result of ana rt ifact , a nd is not a reflection of the correct an sw er. Avoiding th e emba rra ssmentof publishing a retra ct ion is one reason for t he care t ha t people ta ke in performingexperiments a nd in interpreting th e results.

Another ethical issue is th e proper cita tion of th e sources of informa tion you use forany scientific writing. You should always properly reference the authors of papers orbooks you consult . I t a lso means tha t you should cite th e inventors of methods t ha tyou use for your experiment s. If y ou do not, you a re, in effect, cla iming credit for workperformed by others.

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General Information: Keeping a Laboratory Notebook

All student s w ill be required to ma inta in a labora tory n otebook. The notebook will beused for the recording of laboratory data and calculations, and will be criticallyimporta nt for w riting your lab reports.

The purpose of a laboratory notebook is to allow anyone with some biochemicalknowledge to understand exactly w ha t y ou did. You need to record t he informat ion insuf f icient de ta i l so a s to be a ble to repea t i t , an d you must be able to understa ndexa ctly wh a t y our results w ere. You w ill need good notes to be able to write your la breports ; in a ddition, a s your un dersta nding of biochemistry improves, your n otebookma y allow you to figure out why some par ts of your experiment s did not work asexpected.

Compa nies th a t perform resea rch requir e their employees to keep proper notebooks.In these companies, company policy dictates that any work not recorded in thenotebook w a s never a ctua lly perform ed. As a result , th e w ork must be repeated,

w hich tend s t o ha ve deleterious effects on t he car eer opportunit ies of t he employeesinvolved. In cases of disputes a s t o priority , notebook dat es ar e sometimes used t oindica te exa ct ly w hen a n experiment wa s performed. Ownership of patent s (a nd insome ca ses la rge a mounts of money) ca n t herefore be critica lly dependent on keepinga proper n otebook. In str uction in keeping la borat ory notebooks is t herefore a ma jorpart of most la bora tory courses.

In your notebook, ea ch experiment sh ould begin w ith a title, a date, and a s t a t ementof th e objectiveof th e plan ned w ork. You should a lso record exactly what you didat each step (being sure to mention any th ing tha t you did tha t dif fered from theinforma tion in th e Manua l). In a ddition, you should record a ny n umerica l informa tion,such as the weights of reagents used, absorbance readings, enzyme activities, protein

concentrations, and buffer concentrations.

Most experiments will extend over several days, and over several pages in yournotebook. To allow y ou to keep tr a ck of w ha t you ha ve done, you sh ould include t heda y s da te a t the t op of each page . Inc luding sub-t i t les for ea ch pa ge may ma ke i teasier to keep tr a ck of wh a t you did at each step.

Everything you do should be recorded directly int o your la b notebook in pen. If youma ke a m ista ke, dra w a line th rough it , an d wr ite the correction next to the mistake.(It may turn out that the original information was correct after all, so do notoblitera te th e origina l informa tion by erasin g it , or by removing the page from yournotebook.) Any calculations performed should be written directly into your book. Hard

copies of work done on a comput er an d printout s from labora tory inst rument s shouldbe taped directly into your lab notebook.

Writing importa nt informa tion on scra p paper, and th en recording it in y our n otebooklater is not acceptable. If you are writing something while in the laboratory,you should be writing it directly into your notebook.

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At ea ch st ep in your experiment (af ter ea ch assa y or measurement ), in ad dit ion tothe results , record your th oughts regarding t he experiment a nd h ow you think i t isgoing. Record your mistakes, and your attempts to rectify them. Record theca lculat ions involved in a ny t ype of dat a a na lysis , a s well a s expla na t ions for bothw ha t you did an d w ha t you think i t mea ns. A resea rch project is a journey into theunknown ; your labora tory notebook is usua lly your only guide th rough th e forests ofuncerta inty.

It is a lso a good idea to look over your notebook periodically dur ing t he semester, a ndma ke notes of th ings th a t you do not understand, so th at you can ask quest ionsbefore the lab r eport s a re due.

Do not sa y w ell , I wil l remember w ha t t his means ; instea d, w r i t e i t d ow n! D o notsay I w ill remember wha t I wa s thinking while I did this experiment; instea d, w r i t e i t d own! I f y ou use your lab n otebook properly, you w ill find t ha t w rit ing y our la breport s is much easier, a nd y ou will be developing good ha bits for the fut ure.

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General Information: Laboratory Reports

The la bora tory reports are ma jor w rit t en assignment s, due a t int ervals during th esemester. The la borat ory report s should be wr itt en in t he form of a scient ific paper.To help you lea rn to w rite a scientific pa per correctly, th e labora tory r eports w ill bedue in sections, w ith each report building on th e previous one. Each report shouldcontain all of the information from the previous report, plus all of the newwork. You should incorporate the instructors suggestions, using these comments toguide you in th e genera tion of the new sections. Note tha t t he lat er laborat ory report sw ill be gra ded more stringently t ha n ea rlier ones: you are expected t o learn from yourmistakes!

The laboratory reports should contain the following sections:

Laboratory Report 1: Title P a geMat erials a nd Methods

Result s/Discus sion

ReferencesAcknowledgments

Appendix(La bora tory R eport 1 covers E xperiment 2)

Laboratory Report 2: Title P a geIntroductionMat erials a nd MethodsDiscussion

ResultsReferencesAcknowledgmentsAppendix

(La borat ory Report 2 covers E xperiment s 2 and 3)

Laboratory Report 3: Title P a ge

IntroductionAbstrac tMat erials a nd MethodsResults

Discussion

ReferencesAcknowledgmentsAppendix

(La borat ory Report 3 covers E xperiment s 2, 3, an d 4)

The Final Laboratory Report is a revision of Report 3, and thus also coversExperiment s 2, 3, a nd 4. Note th a t t he results from Experiment 5 ar e not included ina ny of the laborat ory reports; instea d, you w ill report y our results from E xperiment 5in a poster.

All of the laboratory reports are expected to be well formatted, word-processeddocuments, written in standard scientific American English. The use of spell-checkersa nd gra mma r checkers is strongly recommended.

(Note: th e Appendix does not ha ve to be as n eat ly format ted a s t he rest of the report ,an d, if necessary, may be ha ndw ritten.)

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Writing Laboratory Reports

In scientific resear ch, results a re report ed to th e world in th e form of scient ific paperspublished in the peer-reviewed scientific literature. These papers are not onlyimporta nt in disseminat ing th e results of th e resea rch, but a re crit ica l for essentiallya ll a spects of ca reer a dva ncement for the scient ists involved. Learning to write aproper scientific paper is therefore an important part of the education of allscientists.

S cient ific pa pers a re expected t o be wr itt en in a w ell-defined forma t. The overa llformat is generally similar in all journals, although the specific details varysomewha t. In this class, the laborat ory reports should be in t he form of a paper in t heJournal of B iological Chem istry. Looking for papers in the J our nal of B iologicalChemistryto use a s exa mples is strongly recommended. (Note th a t t he forma tt ingtha t you should a t t empt t o emula t e applies to content; you do not need to spendtime genera ting th e specific page la yout of a J our nal of Biological Chemi str ypaper.The preferred pa ge layout for la b report submission ha s t he body of your pa per in

double-spa ced text .)

Ma ny scient ists ha ve th eir ow n preferred w a ys of writ in g pa pers. Most scient ists ,however, use an it erat ive process of wr iting, in wh ich t hey w rite the paper, and th enrewr ite i t severa l t imes before submit t in g the paper to th e journal for review an d(hopefully) publica tion. In a ddition, most pa pers a re writ ten in a n order tha t devia tesfrom the f ina l forma t . A common procedure is to writ e th e Methods sect ion f irst ,followed by the Results section. The Methods section is a simple description ofprocedures a nd ca n be w rit ten before the experimenta l results ha ve been a na lyzed.The Results section contains the observations that constitute the study to bepublished. Once these sections a re w ritt en, most people write a n in complete dr a ft ofth e Discussion section t ha t expla ins th e results in t he context of the pa per.

After the Results section is written, and some thought put into interpreting theresults, most people write the Introduction. When writing your Introduction, youshould th ink of th e In tr oduction a s a n episode of J eopardy : the Results a re t hea nsw ers, an d now it is necessa ry t o come up wit h corresponding q uestions. You do notneed to write th e quest ions in t he form of a q uest ion, but y ou should think a boutra ising quest ions in th e rea ders mind th at you will then a nsw er in the Results a ndDiscussion sections.

After writing the Introduction, you should then look at how you have writtenInt roduct ion, and rewrit e the Results sect ion to more clea rly a nsw er the quest ionsra ised in th e Intr oduction, an d then w rite the Discussion to interpret a nd clarify the

a nsw ers. When properly done, each rewr ite acts a s a n impetus for th e rew rite of adifferent section, until a ll of th e sections fit t ogether int o a coherent st ory.

Fina lly , a f ter a l l of the other sect ions ha ve been wr it ten, you can wr ite the a bstra ct ,by extra ct ing t he most importa nt informa tion from each sect ion an d combining th einforma tion into a single para gra ph.

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You sh ould keep these genera l concepts for w riting a paper in m ind w hile consideringthe content of each section. The content of each section of a scientific paper isdiscussed below. (Remember th a t you will not w rite t he paper in t his order.)

Title Page: This sh ould include the t it le of your r eport , th e au th ors na me (i .e. yourname), your lab partners name(s), and your address (your e-mail address is

sufficient).

Abstract: This should be a briefversion of the entire paper. It therefore shouldinclude a br ief int roduction, met hods, resu lts , and d iscussion, expressed in ~ 200words. This truncation is normally achieved in part by greatly abbreviating themethods portion, unless the methods involved are novel or are crucial tounderstanding the findings presented.

Thousands of papers are published every week. Most literature database searchengines include the tit le and a bstra ct , but do not include the remainder of the paper.In w riting the a bstra ct , remember tha t t he vast m a jority of rea ders probably w ill notread the paper, because they lack the time. Therefore, in order to present yourinforma t ion to the la rgest possible a udience , you need to have a n a bst ra ct t ha t isclear ly wr i t ten, tha t is unders ta nda ble wi th out ha ving to read the paper , and t ha tcont a ins a ll of th e relevant findings from t he paper.

The abst ra ct should end w ith t he overall conclusions f rom t he paper ; once aga in ,th is is import a nt because you want people to know what you have discovered. Yourjob/gra nt fun din g/promot ions/fa me a nd f ortun e/a bilit y t o do more experimen t s/a bilit yto retir e to the exotic locale of your choice ma y depend on ha ving people und erst a ndw ha t y ou ha ve done. (This a pplies to th e entire paper, but t he a bstra ct t ends to be atlea st skimm ed by va st n umbers of people w ho will never rea d t he paper.)

Introduction: This section should include background information setting up thescientific problem you are attempting to address and the overall goal of theexperiment s you performed. Wha t is th e hypoth esis you a re testin g? Wha t dir ectlyrelevant information is necessary to understand this hypothesis and why is itimporta nt? Wha t is not known t ha t you hope to a ddress? Wha t a re you pla nning t oa tt empt t o accomplish? (Very br iefly) How did y ou a ccomplish t his?

In w riting a n introduction, you are at tempting to orient t he rea ders, so tha t th ey willknow wh a t t o consider as they rea d t he rest of the paper. This means tha t y ou shouldcarefully consider whether you are presenting information that is irrelevant ormislea ding. If you discuss an issue rela ted to your protein in the introduction, th ereader will expect you to address that issue in the remainder of your paper. In

a ddition, a fter ha ving read y our intr oduction, th e reader should ha ve an a ppreciat ionof th e qu estions you w ere a tt empting to a ddress w ith your experiment s and w hythese questions are important. If someone can read your introduction withoutwa nt ing t o rea d th e res t o f your paper to find the a nsw er to the burning quest ionstha t y ou ra ised, you have not w ritt en your introduction properly!

Methods: This section sh ould be a concisesumma ry of w ha t you did. It sh ould includeenough deta il so th a t a ny rea sonably int elligent biochemist could repeat your work,

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but not a minute-by-minute recitation of the hours you spent performing theexperiment. One common mist a ke is t o include informat ion th a t belongs in theResults section; t he Meth ods section is for methods. For exam ple, a description of aprotein ass a y should describe th e procedure used, but genera lly should not include alist of the sa mples measured in t he assa y. On th e other ha nd, a common mista ke isto fa i l to include some methods, such as the techn iques used to an alyze the dat aobta ined during the study.

When most people rea d a paper, th ey tend t o skipth e Meth ods sect ion unless th eyneed to know exa ct ly how a n experiment w a s performed. This means th at they w illnot read the Methods unless they do not believe your description in the Resultssection, or because they work in the field and want to see if you used a noveltechnique. Because many people skip the Methods section, the Methods sectionshould only be a description of t he met hods used. With th e possible exception of one-time events such a s plasm id const ructions, it is ra rely a good idea to include results inth e Methods section. If you do include results in t he Methods section, th ese resultsshould be a t least summa rized in the Results section a lso.

The Methods sect ion should a lso conta in t he source of th e importa nt reagent s a ndident ifying informat ion for a ny equipment used. B eca use resea rch reagents of highqua lity a re ava ilable from ma ny vendors, th e precise source of most rea gents is muchless importa nt th a n i t once w a s. I t is common pract ice, however, t o st a te in t heMethods s ection t ha t , for example, th e ADP -glucose pyrophosphoryla se expressionvector w a s a generous gift of Dr. C . Meyer.

Results: This section sh ould be a d escription of wha t y ou did in words, illustra tedwith f igures and ta bles. I t is not enough m erely t o have severa l figures; you need toexplain what each figure means. Try to avoid merely listing results in the text;instead, expla in t he findings a nd briefly fit t hem int o the overa ll context of th e paper.

For each set of experiment s, you need to consider t he follow ing q uestions: Wha t a reyou doing? Why a nd h ow a re you doing it? Wha t w a s th e rat iona le for t he methods youemployed? Wha t is t he point of th e experiment you a re a bout to describe? Wha tstra tegy a re you using to address th e experimenta l question you are asking?

None of your a nsw ers to the a bove quest ions sh ould be length y, but you do need toconsider these questions in w riting your report . I t ma y be t ota lly obvious t o you w hyyou performed your brilliant experiment, bu t unless you explain th e purpose an dra tionale behind th e experiment, your flaw less reasoning ma y n ot be obvious t o yourreaders.

Remember t ha t you are t elling a story t o people wh o have not done the experiment s.You cannot assume that the reader will know what you are doing and why. Ina ddit ion, you a re tell ing a story t ha t people w il l be predisposed to disbelieve. Youth erefore need to present your informa tion a s clea rly a s possible. If you do so, peoplewill (a t w orst) understa nd w ha t t hey ar e cr i t icizing, an d (a t best) see tha t you ha veput enough thought and effort into your work as to make it likely that you aretrustworthy.

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Wha t da ta do you need to report ? Do not report da ta merely beca use i t is ava ilable.In stea d, report da ta to ma ke a point . You are trying to tell a factual story. Thism ea n s t h a t y ou cannot l ie to your rea ders. On the oth er ha nd, i f you perform anirrelevan t experiment , reporting t he results ma y be confusing . For exam ple, if youperform five SD S-P AG E electrophoresis experiments t ha t sh ow essentia lly the sa meresults, you do not need t o include the r esults of each individua l gel.

In reporting the results of an experiment that yielded numerical data, it is poorwr iting technique to simply list in th e text t he sam e values listed in a t a ble or shownin a gr a ph. The raw numbers a re meaningless unless put int o cont ext. In oth er words,cite in the text only the impor tantnumbers, and explain whythese values areimportant .

For reportin g numbers in the t ext, convert th e numbers to reasona ble values. Anu mb er s uch a s 0.0014567 mg/l is not reasonable for two reasons: 1) converting theva lue to 1.4567 mg/ml results in a num ber th a t is m uch easier to rea d, a nd 2) th enum ber of significa nt figures reported seems excessive (unless y ou really believeth a t your experiment w a s a ccura te to five significa nt figures).

As a n example, you will be w riting a description of LDH purifica tion an d LD H enzym ea ssa ys in your first Results section. You should consider th e follow ing in w riting t hissection.

P urifica tion: Why did you perform t he purifica tion? Wha t str a tegy d id you employ forthe purification? Why did you use the steps you used and not others? During thepurifica tion, wh a t step resulted in t he great est purifica tion? When did you observe theLD H t o elute from th e column? Wa s th is expected, un expected, or did you ha ve noba sis for ma king a predict ion? Is th ere a f igure you could genera te to clar ify yourresults? (Is a figure necessa ry t o clar ify your results?) B a sed on your da ta , wa s your

purifica tion successful or un successful? Why? Do you ha ve an y da ta oth er th a n fold-purification to indicate whether your purification was successful? How did yourpurifica tion compar e to literat ure va lues obta ined for simila r proteins?

Characterization: Scientific research involves intelligentobservat ion. In oth er words,you need to look at your dat a crit ica lly, and t o a tt empt t o understa nd everything it istelling you. Why did you run gel filtra tion chromat ography or SD S P AG E on yourprotein sam ple? Simply looking at a n SD S P AG E or gel f il t ra t ion experiment a s amethod for determining the molecular weight of your protein may result in yourmissing importa nt informa tion a bout y our protein. If you ra n t hese experiment s on asa mple th a t you believe to be high ly purified, you should examine th e results a ndcompare them to what you would expect to see for a completely homogeneous

prepa ra tion (in other words, to a prepar a tion conta ining zero cont a mina nt s). Thus, inexamining a gel filtration chromatogram, do you see any unexpected peaks? Forexample, if you expect to ha ve a single, monomeric protein, a nd you see tw o peaks ona chroma togra m, you need to f igure out wh ich peak is your protein an d w hich is acont a mina nt , how much of th e conta minat ing ma teria l is present , where i t cam efrom (especially if it w a s not t here previously), a nd w heth er it is necessa ry t o performa dditiona l purificat ion st eps. It is possible th a t t he second pea k is a loosely a ssociat edprotein th a t int eracts w ith your protein; in wh ich case, you ma y be lea rning useful

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informa tion about your protein. Do you see evidence on a n S DS P AG E on t he sa mesa mple for similar cont a mina nt s? Why or wh y not?

Enzyme a ssay : what can you learn from each enzyme assay? (If the answer isnothing, is it w orth including these results in the paper?) H ow do you know th a t t heassay results are valid? What assumptions are you making about the enzyme

rea ction a ctua lly occurrin g in th e reaction tube? Are th ese a ssumpt ions likely to becorrect for each a ssa y? Are th ese assum ptions l ikely t o be correct for some a ssa ysbut not for others? Wha t contr ols did you run t o ensure tha t t he results w ere a t leastpotentia lly mea ningful?

In some ca ses, the a nsw ers to the a bove questions do not n eed t o be stat ed explicit ly.However, you always need to consider the answers before writing the paper.Knowingly incorpora ting t he results of a fla wed experiment in a paper is a good w a y tolose grant funding or become unemployed, and may result in your finding yourself incourt defending yourself in a law suit or in a cr iminal t r ia l . This does not mea n t ha texperiments t ha t la ter tu rn out to be less informa tive tha n you would like are useless,but merely mean s tha t you should look ca refully at your dat a , and t ry to understa ndthe va lidity of each experiment before mentioning it in a wr itten document.

Discussion: This section should begin w ith a brief summa ry of your results, a nd a nexplana tion of wh a t t hey mea n. Wha t w ere you hoping to a ccomplish? Wha t did y oudiscover a s a result of your experiments? Which of your result s a re interest ing? Wha tca n you say a bout your hypotheses now t ha t you have add itiona l dat a ? Wha t did youexpect t o see? Did y ou see wh a t y ou expected? Did you find sur prising result s?

At leas t in pa rt , the Discussion section should be th e section in w hich you a nsw er thequestions you ra ised in the In troduction. Sometimes the answ er is tha t your originalhypothesis turned out to be flaw ed; in t his ca se, you point out h ow t he da ta indicat e

th e f la ws , an d propose a bril l ia nt new hypothesis to account for your observa t ions.Sometimes your origina l hypothesis is supported by th e dat a , in wh ich case you pointout how y our origina l brilliant concept predicted your results.

You sh ould end your discussion section w ith your conclusions. D id y our experimenta chieve your goals? How a re your results going t o cha nge th e world?

Figures and figure legends: In w rit ing a paper , f igures ca n be extremely useful.They a re ra rely, however, self-explan a tory. This mea ns t ha t y ou need t o refer to th efigure in the text. In a ddit ion, you need to include some releva nt informa tion in th efigure legend, so th a t people simply glancing t hrough th e pa per ca n derive usefulinformation from the figures.

As a n exam ple, in a f igure of a gel, you should indicate t he ident ity of th e sam plesloa ded in the figure legend. If more tha n one ba nd is present in a n importa nt lan e, it isoften a good idea to highlight th e importa nt ban d in some wa y. (Note: in doing so, donot w rite on t he actua l lane; instead, place an a rrow or other ma rker beside the gel, orbeside th e lane.)

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Designing figures requires considera ble th ought. Wha t point a re you try ing to ma kewit h t he f igure? Is t he point necessary? If t he point is a necessary one, how ca n t hefigure be used to ma ke th e point a s clear a s possible? Ca n you design a f igure t opresent more informa tion, or present t he informat ion m ore clearly?

Figure legends can be extremely useful in allowing you to present relevant

informa tion th a t w ould disrupt t he orderly flow of idea s in th e text. The figure legendsa re a lso necessary in cla rifying th e informa tion presented in t he figure.

References: In a ny scholarly endeavor, it is customar y t o give credit t o your sourcesof informa tion. The Reference section a llow s you t o properly credit t he origina tors ofth e informa tion you a re presenting. Where did your int roductory informa tion comefrom? Where did your methods come from? (Note that, unless you invented themethod, you should alw a ys reference th e pa per t ha t first described the w ork.)

Acknowledgments: In sc ient i f ic papers , i t i s customary to tha nk th e agency t ha tfunded th e resea rch. In a ddition, it is polite to a cknowledge gifts of reagents or other

supplies. Note th a t, if you purcha sed the rea gent, t he source of th e reagent sh ould becited in t he Methods section.

Appendix: Fina lly , the report should conta in an a ppendix tha t conta ins your ra wdat a an d th e ca lculat ions tha t you used to reduce your da ta to understa nda ble form.In a real paper, Appendix sections are only included for the description of novelcalcula tions; in th is course, th e Appendix is included so tha t y our la b instr uctor cancorrect your calculation mistakes.

In each section, at tem pt to orga nize the informat ion you a re presenting logically.Scient ific papers a re w ritt en for int elligent people who ha ve not done the experiments

you are describing. If your report is disorgan ized th ey ma y not und ersta nd it. I f you donot write well, the reader will not believe your conclusions. (In the real world, a poorlyw ritt en paper w ill not be published, and you will not get gra nt funding! In this class, ifyou inst ructor does not believe your conclusions, y ou will n ot get a good gr a de.)

The list of qu estions below is design ed to help you wr ite ea ch section of th e reportcorrect ly . Rea ding over th is l ist of quest ions before w rit ing a dra ft of the report isstrongly recommended. Readin g th ese questions a fter writing y our first draft , a ndusing t he quest ions t o guide your revisions is a lso str ongly recommended.

Criteria for J udging Lab Reports:General:

Does it conta in t he required sections?Is it clear ly writt en?Does it use scientific terms properly?Does it use good gra mma r?Are t he w ords spelled correctly ?Are the calculations performed correctly?

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Is it unnecessa rily long?Is t he title mea ningful?Does th e tit le pa ge conta in the a uthors na me and a ddress?Does the title page conta in th e nam e(s) of the a uth ors lab pa rt ners?

Abstract:

Does it int roduce th e overa ll topic?Does it explain th e hypoth esis being tested?Are th e importa nt methods described?Does it rea ch logica l conclusions supported by t he da ta ?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Introduction:Does it give genera l background?Does it point out poorly un derstood or unknown factors relat ed to th e study?Does it ra ise questions?Does it explain th e hypoth esis being tested?Does it discuss th e significa nce of th e work?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Materials and Methods:Could th e experiment s be understood based on the informa tion given?Does it include the source of th e reagent s?Does it include informa tion th a t belongs in th e Results section?Does it describe a ll of th e methods used?Is it excessively long?

Results:Does it expla in th e rat iona le and st ra tegy for the experiments performed?

Does it describe, in words, wh a t w a s done?Does it a nsw er the questions ra ised in th e Intr oduction?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Discussion:Does it summa rize the findings obtained in t he Results section?Does it discuss the expected results?Does it discuss t he unexpected results?Does it a nsw er the questions ra ised in th e Intr oduction?Does it reach conclusions?Does it expla in w hy t he conclusions ar e importa nt ?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Figures:Are th e figures well designed?Do th e figures include informa tive legends?Do th e figures present informat ion useful for und ersta nding t he text?

Tables:Are th e ta bles well designed?

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Do the ta bles present informa tion useful for understa nding t he text?Is th e informa tion in the ta bles redundan t?

Acknowledgments:Are t he sources of funding given credit?

References:Is t he informa tion obta ined from published sources properly referenced?

Appendix:Are the raw da ta a nd th e ca lculat ions included?

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Experiment 1: Introduction to TechniquesExperiment 1AUse of Pipetmen

In biochemist ry, th e a bility t o a ccura tely and r eproducibly mea sure a nd tr a nsfer

sma ll volumes of liquids is critical for obta ining useful results . For volumes less tha n 1ml, the most comm on met hod for meas urin g liquid volumes involves th e use of adevice known as a pipetman. (Note: Pipetman is the brand name of the mostcomm only used of th ese types of pipets; how ever, all of th ese pipettin g devices w orkon similar principles.)

A dra w ing of a pipetma n is sh ow n a t r ight. The devicesyou use ma y n ot look exactly like the one shown . Thepipetm en used in t his cours e come in t hr ee differentty pes: P 1000, P 200, a nd P 20.

P 1000 a re us eful for volumes from 200 to 1000 l.

P 200 a re usefu l for volum es from 20 t o 200 l. P 20a re useful for volumes from 0.5 t o 20 l. Ma ke surethat you are using the correct pipetman for thevolume you need. Also, ma ke sure tha t t he pipetma nis a ctua lly set for t he volume you need by looking inth e volume w indow, and, if necessa ry , turning th evolume control knob unt il the pipetma n d ispla ys t hecorr ect volume (t he pipetmen d o notread your mind;beca use severa l people w il l use the pipets, t hey m a ynot a lwa ys be set a s you expect t hem to be). Do notattempt to set pipetmen for volumes larger thantheir maximum, or for volumes less than zero;doing so will dama ge the pipetman .

All pipetmen use disposable tips (do not pipet liquids without using theappropriate tip, beca use this wi l l conta mina t e the p ipetma n a nd ma y da ma ge it ).When a tt a ching t he tip, make certa in tha t the t ip is the correct type for thepipetman you are using, and that the tip is properly seated on the end of thepipetman.

Try depressing t he plunger. As t he plunger depresses, you w ill feel a sudden increasein resist a nce. This is th e first st op. If you continu e push ing, you will find a pointw here the plunger no longer moves dow nw a rd (th e second stop). When using th e

pipet, depress the plunger to th e first st op, pla ce th e tip into the liquid, a nd in aslow, controlled manner, a llow th e plunger to move upwa rds. (Do not simply let t heplunger go; doing so will cause the liquid to splatter within the tip, resulting inina ccura te volumes an d in conta mina tion of the pipet.)

Now , ta ke the pipetma n (ca rrying t he pipet ted l iquid in th e t ip) to the conta iner towh ich you w ish to a dd liquid. Depress th e plunger to the first , a nd t hen to th e secondstop. If you wa tch carefully, you will note tha t d epressing to th e second st op expels allof the liquid from th e tip. (Actua lly, this is t rue for most a queous solutions. In some

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cases, however, such as for organic solvents, or for solutions containing largea mounts of protein, it is often difficult t o get a ll of th e liquid out of the tip. In t hesecases, it is best t o wet t he tip, by pipett ing t he original solution once, expelling it ,an d th en ta king up the liquid a second t ime.)

Although pipetmen a re tremendously useful, they h a ve a potentia l dra wba ck. If used

improperly, pipetmen will tra nsfer inaccurat e volumes. In addition, pipetmen ma ylose calibr a tion. If used incautiously, t herefore, pipetmen ma y yield misleading oreven totally useless results. Checking the calibration of pipetmen is a simpleprocedure tha t ca n sa ve considerable t ime, energy, a nd r eagents. I n t his experiment ,you w ill lear n how to use pipetmen of va rious sizes and measur e their a ccura cy,precision, and calibration.

Experimental Procedures

Materials

PipetmenP ipet t ipsWaterBalancesWeigh Boats

Methods

For each P ipetm a n you will w a nt to check the a ccura cy a nd precision over t he entirera nge using a t lea st t w o different volumes (for exam ple: if you ar e using a P 1000 youw ill w a nt t o check 300 l a nd 1000 l).

Accuracy i s a measur e of proximity t o the t r ue va lue or th e expected va lue for ameasurement. Precision is a measure of reproducibility. For example, obtainingw eight s of 0.5, 1.0, an d 1.5 gra ms for a P 1000 set for 1000 l w ould be accura te, butnot precise; obt a inin g 0.67, 0.68, an d 0.67 g for t he sa me sett ing w ould be precise, butnot a ccura te .

1. Acquire Pipetmen a nd th e correct size t ips.2. P lace a w eigh boat on the balance and ta re the weight t o zero.3. Dra w up the designa ted volume of deionized w a ter into the pipet tip and dispense it

ont o the weigh boa t. Record the weight of the w a ter add ed.4. Repeat the procedure twice for each volume (yielding a tota l of thr ee w eights for

each volume).

The P 20 uses very sm a ll volumes, which ha ve very sm a ll weights. In order to obtaina ccura te readings w ith t he rela tively low precision ba lan ces ava ilable, you may needto pipet t he volume of w a ter s everal t imes (5 or 10 times is recommen ded) for eachvolume being test ed. For exa mple, pipet 10 l 10 tim es in succession, a nd record t hew eight of the 100 l tota l volume a s one measur ement. Ana lyze the da ta you collecta s described in t he section on da ta reduction (below ).

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Prelab Questions #1B

1. Your lab partner hands you a P200P ipetman set as shown in the diagra m a t r ight .Wha t volume is i t set for? Is this the properP ipetm a n for t his volume? Why (or w hy not)?

2. You ha ve a 0.5 M st ock solution of Tris ba se. How w ould you ma ke 100 ml of 0.03

M Tris base?

3. If you perform a 1:4 dilut ion on 50 mM Tris ba se, wh a t is t he fina l concentr a tion?

4. Is the extinction coefficient for a molecule the same for all wavelengths?

5. You prepare several dilutions of an unknown compound. You measure thea bsorban ce of ea ch solution a t 340 nm u sing a 1 cm cuvette (your results a re listed inth e t a ble below). Wha t is th e extin ction coefficient (in (M cm )-1) of th e compound?

(Hint: assume that each of the individual values contains some degree ofexperimenta l error.)

Concentration(M)

Absorbance at340 nm

2 0.009

4 0.020

8 0.061

16 0.111

32 0.189

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Experiment 1BSpectroscopy and Dilutions

SpectroscopyA spectrophotometer is a n inst rument for mea suring t he a bsorbance of a solut ion.

Absorba nce is a useful qua ntit y. The Beer-La mbert law sta tes tha t :A = clwhere A is th e a bsorban ce of the sample a t a par ticular wa velength, is th eextinction coefficient for t he compound a t th a t w a velength in (M cm )-1, cis the molarconcentra tion of the a bsorbing species, an d lis the pa th length of the solution in cm.Thus, if t he extinction coefficient of a n a bsorbing species is known , th e ab sorban ce ofth e solut ion can be used to ca lcula te t he concent ra t ion of the a bsorbing species insolution. (This a ssumes th a t t he species of interest is th e only ma teria l tha t a bsorbsat the w avelength being measured.)

The above is an explanation of whywe mea sure absorbance: a bsorba nce a llow s us tocalculat e the concentr a tion of compound s in solution. How ever, it does not explain

whata bsorbance is. Another d efinition of a bsorba nce is:

A = log

I0

I

where I0 is the amount of light

entering the sample, and I is theamount of light leaving the sample.Absorbance is therefore a measureof th e portion of the light lea ving th elamp tha t a ctua l ly ma kes i t t o thedetector. A little thought will revealtha t w hen absorban ce = 1, only 10%

of th e l ight is rea ching the detector ;w hen a bsorban ce = 2, only 1% of thelight is rea ching the detector.

The typica l interna l arr a ngement of aSpectrophotometer

Absorbance values greater than 2 are unreliable, because too little light isreaching the detector to allow accurate measurements. When measuringa bsorba nce, note the va lues; i f the rea ding is greater t ha n 2, dilute the sa mple a ndrepeat t he mea surement.

Spectrophotometers measure the decrease in the amount of light reaching the

detector. A spectr ophotometer will interpret fingerprin ts on the optical face of th ecuvette, or air bubbles, or objects floating in your solution as absorbance; youtherefore need to look carefully at your cuvette before putting it into thespectr ophotometer to ma ke sure tha t y our readin g is not subject to these types ofar t i facts .

Cuvet t es ar e usually sq ua re objects 1 cm a cross (a s shown in th e above figure). Insome cases, the liquid reservoir is not sq ua re; in those ca ses, make sure th a t t he 1 cmdimension is a ligned w ith t he light path (note the orientat ion in the dia gra m a bove.)

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Some cuvettes a re designed for visible light only. When t he spectr ophotometer is setfor ultr a violet wa velengt hs (wa velengt hs of 340 nm or less) make sure that yourcuvette does not have a large absorbance when it contains only water.

The t erm spectr oscopy comes from t he w ord spectr um w hich originally referred tothe multiple colors of light apparent in an analysis of white light using a prism.Spectroscopy therefore implies the use of multiple wavelengths of light.Spectrophotometers ha ve the a bil ity to specif ica lly mea sure a bsorba nce at specif icwavelengths. The most commonly used method to allow this involves amonochroma tor, a device (either a prism, or m ore commonly, a diffraction gra ting)tha t splits th e incident light into its component w a velengths, a nd a llow s only light ofth e desired wa velengt h to reach th e sample. The abil i ty t o mea sure absorba nce a tdifferent wavelengths is very useful, because the extinction coefficient of acompound varies with wavelength. In a ddition, the a bsorba nce spectrum of acompound can vary dramatically depending on the chemical composition of thecompound, and depending on the environment (such as the solvent) around the

compound.

The graph at right showsthe absorbance spectrumof a protein. The proteinhas a s t rong absorbancepeak near 280 nm, butexhibits very littleabsorbance at longerwavelengths. For thisprotein, the onlychromophores (chemical

groups with in a compoundth a t a bsorb light) ar e thearomatic amino acidstryptophan and tyrosine.For many proteins, these two residues are the only chromophores; becausetry ptopha n a nd t yrosine only a bsorb in th e ultra violet portion of th e spectru m, suchproteins a re colorless m olecules. C olored proteins , such a s hemoglobin, exhibit t heircolor due to chr omophores (heme, in t he case of hemoglobin) tha t a bsorb in t he visibleport ion of the spectr um.

The extinction coefficient of a molecule at a given w a velengt h ca n be calculat ed usingth e B eer-La mbert equa t ion from absorban ce measur ements for solut ions of know n

concentration.

DilutionsMany solutions used in biochemistry are prepared by the dilution of a moreconcentra ted st ock solution. I n prepar ing t o make a dilution (or series of dilutions),you need to consider th e goa l of th e procedur e. This mea ns t ha t y ou need t o consider

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both t he desired fina l concentra tion an d required volume of the diluted ma teria l. Asimple equa tion a llow s th e dilution to be ca lcula ted rea dily:

C 1V1 = C2V2

where C1

is the concentra tion of th e init ia l solution; V1

is th e volume of th e init ia lsolut ion ava ilable to be used for dilut ion (th is may n ot be the tota l volume of theinitial solution, a nd inst ead ma y be a sm a ll fraction of the initial solution), C2 i s t h edesired final concentration, and V2 is th e desired fina l volume.

In m ost ca ses, the initia l concentra tion an d th e fina l concent ra tion ar e either knownor ar e chosen in order to w ork correctly in t he experiment b eing plan ned. The fina lvolume is usua lly an a mount th a t is chosen based on the a mount required for a givenexperiment. This mean s t ha t a t least thr ee of th e required t erms a re either known orcan be chosen by t he experimenter.

Let us consider a n example. You a re set t ing up a sta nda rd curve. You ha ve a st ock

solution of 1000 g/ml B S A, a nd for one of th e point s on t he curve, y ou w a nt 200 l of20 g/ml. In this ca se, C 1 = 1000 g/ml; C2 = 20 g/ml, a nd V2 = 200 l. This lea ves V 1as t he unknow n va lue (i .e. how m uch of th e stock solut ion mus t b e dilut ed t o 200 lfina l volume to yield th e desired concentra tion). Rearra nging th e dilution equat iongives:

V1= V

2 C1

C2

a nd th erefore ( )4 l = 200 l20 g/ml

1000 g/ml

Thu s, y ou need t o dilut e 4 l of th e st ock solut ion t o a fina l volume of 200 l (i .e. byadding 196 l).

If, in t he exam ple, you w ished t o ma ke a solut ion of 1 g/ml, th e sa me equa tion w ouldind icat e th a t you n eed 0.2 l of t he 1000 g/ml s t ock solut ion for 200 l of th e fina ldilut ed sa mple. This is a problem: 0.2 l is very difficult t o measu re a ccura tely. Youha ve two choices: cha nge the f inal volume (i .e. if V2 is larger, then V1 must a lsoincrease), or perform serial dilutions (i.e. instea d of diluting th e stock solution by afa ctor of 1000 in one step, dilut e the st ock solut ion, a nd t hen m a ke a furt her dilut ionof the diluted stock).

In m an y cases, wh ile the fina l concentrationis importa nt, th e fina l volumeis not (a s inth e previous para gra ph). In t hese ca ses, do w ha t w a s explained in th is exam ple: use aconvenient dilution: a dilution that involves volumes that are easily pipetted.P ipett ing 1.3333 l is usua lly less a ccura te t ha n pipettin g 4 l, both because 4 l is a

la rger volume, a nd beca use it is d ifficult t o set t he pipet for 1.3333 l. In t his ca se, 4l is a conven ient volume, w hile 1.3333 l is n ot.

In some ca ses, you may not know t he a ctua l sta r t ing concentr a t ion. I f , for example,you need to measure th e enzyme act ivity in a sam ple, a nd you f ind t ha t t he act ivityis too high to measure a ccura tely, you w ill need to dilute the sta rting m a terial. Sinceyou dont know th e actua l sta r t ing concentr a t ion, a l l you know is the concentr at ionratio between starting and final solutions. As long as you keep track of the

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concentration ratio in all of your dilutions, you can easily determine the enzymea ctivity in th e initial solution, even t hough you ca nnot mea sure it directly.

Concentra t ion ra t ios ar e frequently of considerable va lue. For example, you h a ve ast ock solution of buffer th a t conta ins 450 mM Tris-H C l, 10 mM E DTA, a nd 500 mMNa Cl. You a ctua lly w ish to use a final concentra tion of 45 mM Tris-HC l, 1 mMED TA, an d 50 mM Na Cl. In each case th e concentr a t ion of th e f inal buffer is one-tent h t ha t of th e origina l. Simply performing a 1:10 dilution of th e stock solution th engives th e appropria te fina l concent ra tion of each component . The st ock solution ofbuffer is ty pica lly called a 10x stock, beca use it is ten-times m ore concent ra ted t ha nth e fina l, useful buffer.

Note, in the previous paragraph, the 1:10 dilution. The description uses thechemistry convent ion for th is term , wh ich w ill be used th roughout t his course. The1:10 dilution mentioned is performed by ta king one part of the initia l solution, a nda dding nine par ts of solvent (usua lly wa ter). This results in a fina l concentra tion tha tis ten-fold low er tha n t he origina l.

In t his experiment, you will lea rn h ow to prepar e solutions using dilutions, an d learnhow t o use a spectr ophotometer.

Experimental Procedures

Materials

PipetmenP ipet t ips

Para f i lmWater1.0 M Cu SO4 solution

U V cuvet tesVis cuvett esQuart z cuvet tesUnknown C uSO4 solution

10 mM N-a cety l-tr yptopha na mide solution

Simple Dilutions-11. Set t he spectrophotometer to wa velength 700 nm a nd blan k aga inst a ir .2. P repar e 1 ml of th e follow ing dilut ions of the CuS O4 solution using w a ter: 1:2,

1:5, 1:10, 1:50, and 1:100.3. Determine th e A700 for each dilution.

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Simple Dilutions-24. Assuming the C uSO4 solution is a 5X stock, prepa re th e follow ing solutions:

0.5X, 1X, 2X.5. Determine th e A700 for each dilution.

Serial Dilutions6. Prepare the following solutions of the CuSO4 solution using serial dilutions: 1:5,1:25, 1:125, and 1:625

7. Determine th e A700 for each dilution.

Experimental applicationsMeasure the A700 for different dilutions of the unkn own C uSO4 solutions.

Spectroscopy1. P erform a baseline bla nk mea surement for an a bsorba nce spectrum from 250

to 400 nm w ith t he cuvette cham ber empty.2. P erform a bsorba nce sca ns on t he dif ferent types of cuvet tes conta ining only

w a ter. Which cuvett es do you need to use to mea sure a bsorban ce a ccura telyin this wa velength ran ge?

3. Perform an absorbance scan of 200 M N-acetyl-tryptophanamide.4. Prepare dilutions of the N-acetyl-tryptophanamide and measure the

absorba nce at an appropriat e wa velength.

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Experiment 1: Analysis of ResultsUse of PipetmenSet up a ta ble a nd a nsw er the following six questions for each volume mea sured:1. Record the weight you measured for the three tr ia ls .2. Average the three weight s .3. Ca lcula te s t andard devia t ion for your average.1

4. What is the standard deviat ion as a percent of the average value?5. I s the P ipetman accura te a t t h i s volume?6. Is the P ipetman precise a t t his volume?

Do the a ccuracy a nd precision va ry over th e ran ge of volumes for th e Pipetmen?

Simple Dilutions-11. Give the volume of wa ter and CuSO4 solution used for each sa mple dilution.

2. P l ot t h e r es ult s on a n A700vs. Concentra tion of CuS O4 solution graph.

3. Ca lculat e the ext inct ion coefficient for a queous CuSO4 solution.

Simple Dilutions-21. G ive the volume of wa ter an d CuSO 4 solution used for each simple dilution.

2. P lot th e results on a n A700vs. Concentra tion of CuS O4 solution graph.

3. Ca lculat e the ext inct ion coefficient for a queous CuSO4 solution.

Serial Dilutions1. Describe how each seria l dilut ion w as prepared.2. P l ot t h e r es ult s on a n A700vs. Concentra tion of CuS O4 solution graph.

3. Ca lculat e the ext inct ion coefficient for a queous CuSO4 solution.

Absorbance Results

1. Report t he avera ge value an d sta nda rd devia tion for th e extinction coefficient ofCuSO4 a t 700 nm ba sed on your dat a .

2. Est imat e the concentra tion of the unknown C uSO4 solutions based on your dat a .3. Wha t do the results for t he CuS O4 indica te any thing a bout the instrument used

for the measurements (such as the minimum or maximum absorbance valuesthat yield reliable results)?

4. At what wavelength does N-acetyl tryptophanamide exhibit maximalabsorbance?

5. What is the extinction coefficient for N-acetyl tryptophanamide at thiswavelength?

1Sta ndard devia t ion =xi x( )

2

i =1

n

n 1

where xi is each data point , x i s the average va lue , and nis

the number o f measur ements . Most ca lcu la to rs and a l l spreadshee t s wi l l ca lcu la te s t a nda rddevia t ion . No te , how ever , tha t some spreadsh eet s a l so ha ve a lgor i thms fo r popula t ion s t a nda rddevia t ion (in w h ich the denomina tor i s n r a t h e r t h a n n1) . The use of popula t ion standarddeviation is inappropriate for these data. In Excel, the correct function is STDEV(cell range)

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Experiment 2The Art and Science of Protein Purification

When attempting to understand how a protein works, it is usually necessary to

isola te th e protein from oth er proteins t ha t a re present in th e tissue. This a llow s youto study the protein with some assurance that the results reflect the protein ofinterest a nd a re not due to other m olecules th at were originally present in th e tissue.Protein purification is therefore a commonly used biochemical technique.

Most proteins a re fa irly la rge molecules. They a re sma ller t ha n D NA molecules, butthey are tremendously large when compared to the molecules typical organicchemists a re concerned w ith . The t hree-dimensiona l str ucture of most proteins is aconsequence of many relatively weak non-covalent interactions. Disrupting thisth ree-dimensiona l stru cture, on w hich th e function of the protein depends, is th ereforea relatively easy process. Conversely, preventing the loss of the non-covalentstr ucture (a nd sometimes th e covalent st ructure) is frequently d ifficult.

Disrupting cellular structure is required to release the proteins from the cell.However, the process has two side effects that may damage proteins: 1) celldisruption typica lly involves shearing forces and heat , both of w hich can da ma geproteins, and 2) cells normally contain proteases (enzymes that hydrolyze otherproteins). In most cells, proteases a re carefully cont rolled; how ever, disruption of thecell usua lly a lso relea ses the proteases from th eir contr ol systems, a nd ma y a llow thecleava ge of t he protein of interest.

P urifica tion of proteins involves t a king a dva nt a ge of sometimes-subt le differencesbetween th e protein of interest a nd t he rema ining proteins present in th e mixture.B eca use proteins a re a ll polymers of the sa me tw enty a mino acids, the differences inproperties tend to be fairly sma ll.

In most cases, current underst a nding of protein structura l properties is insufficient toa llow a purifica tion meth od to be generat ed theoretica lly. The Art in th e title of th issection reflects t he fa ct th a t d evelopment of most protein purificat ion procedures is ama t t er of tr ia l a nd error. The ta ble below lists s ome of t he genera l properties ofproteins th a t can be useful for protein purif ica t ion, and some of the methods tha tta ke adva nt a ge of th ese properties. Ea ch of these genera l methods w ill be discussedin some deta il below . Note th a t for a ny given protein, only some of these methods w illbe useful, and therefore protein purification schemes vary widely.

Property TechniqueSolubility Ammonium sulfa t e precipit a t ionC ha rge Ion-excha nge chroma t ogra phyH ydrophobicit y H ydrophobic intera ct ion chroma togra phyS ize G el-filt ra t ion chroma togra phyFunct ion Affinit y chroma togra phyS t a bilit y H ea t -t rea t ment , pH t rea t ment

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Ammonium sulfate precipitationIn many cases, cell lysates can be loaded directly onto chromatography columns.However, in some ca ses oth er molecules present in t he lysa te int erfere w ith binding ofthe protein to the resin. In addition, some resins (especially affinity resins andsepharose-based resins) are fairly expensive; loading crude cell lysates on thesecolum ns ma y result in binding of cellular ma teria l (e.g. lipids and DNA) that are

difficult to remove, and which may damage the column. As a result, purificationmethods often begin w ith one of several possible simple techniq ues th a t remove atlea st some of these unw a nt ed ma teria ls prior t o using a n expensive column.

One of th e most commonly used crude purificat ion techniques involves th e use ofdifferential solubility. Proteins precipitate with increasing ammonium sulfateconcentra tions, w ith most proteins precipita tin g somewh ere betw een 10% a nd 60%a mmonium sulfat e. (The percent a ges are relat ive to a sa tur a ted solution, which ha sa concentra tion of a bout 4 M; thu s most proteins precipita te betw een 0.4 M a nd 2.4M.) This can a llow a simple, par tia l, purifica tion of a protein; if t he protein of int erestprecipita tes a t 40%a mmonium sulfat e, ma ny other proteins w ill rema in in solution,a s w ill ma ny other non-protein molecules.

Most proteins are not damaged by ammonium sulfate precipitation, and can beresuspended in a sma ll volume of buffer. Ammonium sulfa te precipita tion results in ahigh sa lt concentra tion in th e protein solution; this ma y be a dva nt a geous (if th eint ended next st ep is hyd rophobic int era ction chroma togra phy), or deleterious (if thenext st ep is ion exchan ge chroma togra phy).

When necessa ry, t w o methods are frequently used to remove the sa lt . One method isgel filtration chromatography (discussed briefly below, and in more detail inExperiment 3D). Another frequently used method is dialysis.

DialysisDia lysis involves placing th e protein solution in a semi-permeable membrane, and placing the membrane in ala rge cont a iner of buffer. Sm a ll molecules (such a s sa ltions) pa ss t hrough t he dia lysis membra ne (moving fromhigh concentration to low concentration), while largemolecules are unable to cross the membrane. Dialysismembranes come in a variety of pore sizes, and areth erefore useful for removing a va riety of different sized solutes. In principle, dia lysiscould allow separa tion of la rge proteins from sma ll ones; in practice, however, th epores in the tubing are insufficiently uniform to allow this technique to be used

effectively.

Chromatographic methodsMost purification methods involve chromatography. Chromatographic methodsinvolve a column of an insoluble ma teria l tha t ca n bind m olecules based on specificproperties common to proteins. The solution containing the mixture of proteins isth en a llow ed to pa ss th rough th e column; th e protein of interest ma y bind (dependingon its properties), w hile a t lea st some impurit ies rema in in solution and lea ve th ecolumn. The procedur e is completed by elut ing (i .e. removing) the proteins t ha t h a ve

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bound to th e column .

An illustration of a chromatographic run is shown above. The initial sample containsfive different proteins (th e different ly colored filled circles). These proteins a re boundto the column fairly tightly. Once elution begins, the proteins begin leaving thecolumn. The graph at the bottom of the diagram shows proteins eluting withincreasing sa lt concentra t ion, in th e man ner tha t w ould occur w ith a n ion excha ngecolumn; otherwise, this dia gra m a pplies t o essent ia l ly a ny type of chromat ogra phicmethod. Note: most columns do not run t his nea tly, especia lly in the beginning of a

purification procedure.

Ion exchange chromatography: Proteins are charged molecules. Electrostaticforces w ill th erefore a llow proteins t o bind t o oth er molecules of opposite cha rge. Ionexcha nge column s a re produced by covalent ly a tt a ching cha rged molecules such asdiethyl-a minoeth yl (DE AE) groups to insoluble carbohydra te resins. In m a ny ca ses,small differences in charge can result in significant separations on ion exchangecolumns. Ion excha nge columns a re typica lly loa ded at low ionic strengt h, and theprotein removed by ra ising th e ionic strength .

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Hydrophobic interaction chromatography:Proteins contain hydrophobic amino acid side-cha ins, some of w hich ar e exposed a t t he surfa ceof the protein. P roteins w ill therefore often bind toother hydrophobic molecules. Hydrophobic

interaction columns are produced by covalentlyattaching hydrophobic molecules such as acylchains or phenyl groups to insoluble carbohydrateresins.

The h ydrophobic effect is st rongest un der h igh ionic strengt h condit ions; h ydrophobicint era ction columns a re th erefore typica lly loaded at high ionic str ength , and t heprotein removed by lowerin g th e ionic strengt h (thus, t hese column s a re th e oppositeof ion exchange columns).

Gel filtration chromatography: In gel filtration

chromatography (also known as size exclusion, gelpermeation, or molecular sieve chromatography),molecules a re separ a ted ba sed on size. Gel filtrationcolumns are made of porous beads packed into acolumn. Different types of beads have somewhatdifferent physical properties that may make themmore appropria te for different proteins. As a solutioncontaining molecules of varying sizes passes through the column, the moleculesdistribute between the inside and outside of the pores depending on their size.Molecules too big for the pores are totally excluded, and elute from the column first.Sm a ller m olecules fit in t he pores, a nd th erefore elute la ter. The elution volume for amolecule is th us inversely relat ed to the size of the molecule. Because sa lts such a sa mmonium sulfat e a re much sma ller tha n proteins, gel filtra tion is frequently used asa method for removing sa lt from a protein solution.

Affinity Chromatography:Ma ny proteins exhibit specific int era ctions w ith oth ermolecules (called liga nds); for exa mple, enzymes mus t ha ve th e a bility to bind t o theirsubstrates, and antibodies exhibit high-affinity interactions with their antigens. In

B e a d

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principle, it is possible to cova lently a tt a ch th e ligan d t o an insoluble resin. A columnproduced from such a r esin is ca lled an a ffinity column .

Affinity chromatography is somewhat less commonly used than the forms ofchromat ogra phy discussed a bove . In ma ny cases , the cova lent a t t a chment of theligand to the column results in st eric cla shes tha t prevent th e protein from binding. In

some cases, although the protein will bind to the affinity resin, the resin is soexpensive tha t other purifica tion methods ar e used inst ead. I n a ddition, ba cteria findmost a ffin it y colum ns t o be a dmira ble growth media, w ith consequ ent deleteriouseffects on th e chroma togra phic usefulness of the columns.

On the other hand, affinity chromatography can be extremely useful for rapidlypurifying proteins. While most proteins cont a in t he charged gr oups th a t ca n int eractw ith ion excha nge resin s, th e specificity of protein-ligand int era ctions mea ns th a tonly a very sma ll fra ction of th e proteins in a cell will bind t o a ny given a ffinity resin.Affinity chroma togra phy can t herefore be a n extremely useful purifica tion technique;in some ca ses, a single affinity chroma togra phy step ma y be the only step necessa ryto completely purify a protein.

Protein Purification StrategiesDeveloping a scheme for purifying a protein remains an empirical process. However,in purifying a new protein, it is sometimes possible to adapt methods used forpurifying similar proteins. In ad dition, pla nning t he procedure before simply try ingdifferent methods can be extremely useful. Examples of this include using anammonium sulfate precipitation step before a hydrophobic interactionchromat ography st ep, beca use t he high concentra tion of a mmonium sulfate t ha tresults from the precipitation will allow the precipitated protein (or the non-precipita ted protein rema ining in solution) to be loaded directly onto t he column. I ncontr a st , a n a mmonium sulfa te precipita ted protein must be dia lyzed (or otherw ise

desa lted) prior to loa ding on a n ion excha nge column . Anoth er frequent ly used schemeinvolves a n inexpensive techn ique such as a mmonium sulfa te precipita tion prior toremove bulk conta mina nt s prior t o run ning a higher resolution but more expensivetechnique such a s a ffinity chromat ogra phy.

As w ith most scient if ic procedures, th e more you know a bout th e protein, a nd t hemore you know a bout protein purifica tion, the more likely it is th a t y ou will be able todesign a successful purifica tion procedure.

BuffersProteins, and especially enzymes, are generally quite sensitive to changes in the

concent ra tions of various solution components. A buffer is a solution th a t is used t ocont rol the propert ies of a process occurring in a n experimen ta l a queous medium . Theterm buffer is related to the ability of these solutions to resist changes in thehyd rogen ion concentra tion, but buffers a lso cont a in other molecules, and a re used toattempt to influence the ionic strength, the activity of proteases, and otherpara meters of the experiment in a ddition to the h ydrogen ion concent ra tion.

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In a ny biochemist ry experiment , the buffer component s must be chosen based onth eir effect on t he experiment. I dea l buffer component s cont rol pH a nd ionic strengt hw ithout intera cting in oth er wa ys w ith th e system being studied. For example, w hilephospha t e is a common physiological buffer, it m a y not be a ppropriat e for somebiochemical experiments, especially if phosphat e is a subst ra te or product of t hereaction being studied. In ad dition, some proteins int era ct poorly w ith some buffercomponents (a fa ct usua lly discovered by t ria l a nd err or). As a n exa mple, Tris is lessth a n ideal beca use of its high pKaa nd th e la rge cha nge in pKath a t i t exhibits uponcha nges in temperat ure. However, Tris is inexpensive, most proteins ar e st a ble inTris buffers, an d Tris rar ely rea cts with biologica l compounds; a s a result , Tris iscommonly used in biochemistr y.

You w ill ha ve seen th e Hend erson-Ha sselbalch equat ion in previous courses. Thisequat ion is useful for calcula ting t he theoretical pH of a solution. It is a lso useful forpredicting w heth er a par ticular compound w ill be useful as a buffer over a given pHrange. However, the Henderson-Hasselbalch equation has its drawbacks. Manybuffers used in biochemica l experiment s deviat e significa nt ly from ideal Henderson-

Ha sselbalch beha vior.pH = pKa+ log

[A ]

[H A ]Henderson-Ha sselbalch equa tion

Because of the commonly observed deviations from ideal behavior, buffers arety pically prepa red by adding th e buffer component s to a cont a iner, adjusting thesolut ion to th e desired pH by a dding a n a cid or a ba se, and then adding sufficientw a ter t o rea ch the expected fina l volume.

For example, a 1 liter of 50 mM Tris-HCl buffer (pH 7.4) with 200 mM sodiumchloride w ould be prepa red by ad din g 50 mm oles of Tris ba se and 200 mmoles ofsodium chloride to a flask a nd a dding w a ter to about 900 ml. HC l would then be add ed

to reduce the pH to 7.4, using a pH meter t o monitor t he chan ging pH, follow ed bya ddition of enough w a ter t o yield a 1 liter fina l volume. (If t he solution cont a ined 1 literbefore ad dition of th e HC l, the fina l volume would be more tha n 1 liter, a nd t hereforethe buffer w ould be less concent ra ted t ha n it should be.)

Note th a t in order t o produce most biochemica lly useful buffers, severa l component smust be added together. This frequently requires careful consideration of thenecessar y d ilutions for each of th e component s.

When performin g experiments w ith proteins, it is ra rely a good idea to dilute th eprotein with water, unless denaturation of the protein is not a concern. Whendena tur a tion is a concern, such a s when performing dilutions for enzyme a ssa ys ,perform t he dilution using a suita ble buffer t o prevent undesirable alt erat ions in t hestr ucture of the protein in solut ion.

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Prelab Questions #2A

1. You ha ve 47 ml of a protein solution. You w a nt to ad d 0.39 g of a mm onium sulfa teper ml. How much a mmonium sulfate do you need to add?

2. How w ould you ma ke up 100 ml of 50 mM Tris-H Cl bu ffer a t pH 7.4, using th e 0.5M Tris ba se stock solution, and a ny other required ma terials?

3. You h a ve 5 ml of 3 mg/ml prot ein solut ion. (This solution is va lua ble so you dontw a nt to w a st e it .) H ow w ould you ma ke up a t lea st 100 l of 0.1 mg/ml using aconvenient dilution (i .e. how m uch buffer a nd how m uch protein solution do you needto ad d, a nd is it possible to pipet th e amount you need accurately)?

4. Wha t do the lett ers PMS F st a nd for? Wha t is P MSF used for?

5. Define superna ta nt an d pellet.

6. What is the purpose of adding ammonium sulfate to the protein solution?

7. Why a re you sa ving t hr ee 0.5 ml aliq uots of your crude homogena te? (Note: youma y need to rea d ah ead to find th e answ er to this question.)

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Experiment 2APurification of Lactate Dehydrogenase from Chicken, Part I

For the rema inder of this course, you w ill use lacta te dehydrogenase (LD H) as t hesub ject of your st udies. LD H (E.C . 1.1.1.27) cat a lyz es th e nicotina mide cofact or-

dependent int erconversion of lacta te a nd pyruva te:

LDH is found in almost all organisms because it plays an important role incarbohydrate metabolism. During conditions in which pyruvate production fromglycolysis exceeds t he a bil i ty of th e cell to meta bolize the pyruva te , LD H converts

the pyruvate to lactate, and thereby regenerates the oxidized NAD required forfurth er glycolysis . LDH a lso a l low s th e conversion of lacta te to pyruva te ; both t hepyruva te a nd N ADH produced can th en be used for other processes.

In most a nima l t issues, LDH is produced from tw o genes, designa ted A and B . The Agene is somewhat more highly expressed in muscle and liver, and its product isreferred to a s th e M isozyme, while the B gene is more highly expressed in hea rt , an dits gene product is referred t o as t he H isozym e. In most species, the gene productsform tet ra meric complexes w ith propert ies th a t va ry somewh a t depending on th erelat ive a mounts of the different isozymes present in t he tetra mer.

LD H a ctivity is rea dily meas ura ble: th e extinction coefficient a t 340 nm of NADH ismuch higher tha n th a t of NAD. If th e only substra tes ad ded to the rea ction are NADa nd la ctat e (or NADH a nd pyruva te), the cha nge in absorban ce at 340 nm should beproport iona l to the cha nge in NADH concent ra tion due to the LD H a ctivity present inthe cuvette.

As mentioned in the introduction to this section, in any protein purification protocol iti s necessary t o t ake a dvan ta ge of the w a y in w hich t he prote in of interest (in th iscase, LDH) differs from the other proteins in the mixture. Most tissues containth ousan ds of proteins; you need to use the propert ies of LD H t o separ a te it from a ll ofth e oth er proteins present.

Most tissues contain proteases (enzymes that degrade other proteins). Avoidingproteolytic da ma ge to your protein can be difficult . Three techniqu es a re commonlyused t o keep proteolysis t o a m inimum : 1) perform th e purifica tion in th e presence ofprotease inh ibitors, 2) perform t he purifica tion a t low tempera tu res (4 C or on ice),a nd 3) perform th e purificat ion in t he minima l am ount of time possible. B eca use youca nnot d o the last of th ese (th e purifica t ion procedure will ta ke more th a n one labperiod), you should keep your sa mple on ice or in th e refrigerat or as much a s possible.

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DefinitionsTris-(hydroxymethyl) aminomethane hydrochloride(Tris-HCl) is a commonly used buffer, and is intended tohelp control th e pH of th e solut ion.

Tris is in expensive an d genera lly inert in biochemica l experiments. These ad va nt a gessomewh a t compensat e for its dra w backs. One draw back is the high pKava lue of 8.1a t 25 C , a va lue tha t is w ell a bove the norma l pH of biochemical buffers. Anotherdra wba ck is the fac t tha t th e pKaof Tr is chan ges by 0.031 pH uni t s per C, a ndth erefore th e pH of a Tris-buffered solution is very t empera tu re-dependent .

2-Mercaptoethanol (-ME) is a reducing agent; it prevents formation of disulfidebonds betw een free cyst eine residues. It a lso inhibit s some proteases.

P henylmet hylsulfonyl fluoride (P MSF ) is a n irr eversible inhibitor of serine protea ses.P MSF is toxic; avoid gett ing P MSF on your skin.

Et hylenediamine t etra a cet ic a cid (ED TA) is a chelat ing a gent ; i t is used t o removemeta l ions from solution. Some protea ses a re dependent on meta l ions (especiallycalcium ions), so ED TA a cts a s a n in hibitor of some proteases.

Reagents

Extraction Buffer:10 mM Tris -H Cl (pH 7.4)1 mM 2-Mercaptoetha nol1 mM P henylmethylsulfonyl fluoride (P MSF )1 mM Et hylenedia mine tetr a a cetic a cid (ED TA)

Ammonium sulfa te (solid)

Ch icken breast muscleCheesecloth50 ml Centrifuge Tubes (four per group)BlenderMicrocent rifu ge Tubes (1.5 ml)P ipet Tips50-ml Falcon Tubes

Procedure

1. Tissue preparation Cut ~ 50 g of muscle t iss ue from th e tissue source (recordth e exact w eight of tissue used). Cut th e tissue into sma ll pieces wit h scalpel or ra zorblad es. Discar d the connective tissue an d fat .

2. Soluble protein extraction Place the minced tissue and 75 ml of coldExt ra ction B uffer in a blender, an d put t he top on the blender. Disrupt t he tissue by

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homogenizing. U se 4 x 30 second bur sts , w ith a t lea st 10 seconds in betw een ea chburst t o a llow t he temperat ure of the homogena te to decrea se.

3. Centrifugation P ut th e homogenized tis sue/buffer m ixtu re int o four pre-chilled50 ml cent rifu ge tu bes (not e: th e mixture will be th e consis ten cy of a th ick milksha ke , so a spa t u la w i l l he lp). B a lance the tubes (i .e. make sure tha t e ach pa i r o f

tubes ha ve the sa me ma ss). Make sure tha t t he tubes a re not too fu l l (you do notwant to spill your mixture inside the rotor). Centrifuges are dangerous andexpensive. Consult your instructor before putting your samples into thecentrifuge! C entrifuge your homogena te for 20 minut es a t 15,000 rpm.

4. Filtration P o u r t h e s up er n a t a n t (i .e. the fluid on top) through two layers ofcheesecloth into a pre-chilled beaker. The cheesecloth removes lipids from thesolution; th e filtr a tion st ep is much easier if you put th e cheesecloth int o a funn el).Discar d t he cell debris pellets (consult your inst ructor a s t o where to put t he pellet soth a t t he smell of rottin g chicken fra gment s w ill not offend people).Measure and record the volume of the supernatant.Save three 0.5 ml aliquots (label th e a liquots Crud e Homogenat e).

5. Ammonium sulfate precipitation Slowly(over a period of ~ 15 minut es) a dd

0.39 gra ms of ammonium sulfat e per ml of superna ta nt to your filtered supernat a nt.It is best t o perform t his st ep in t he cold room on a ma gnetic stirr er (obviously, youneed to put a stir bar into your sample). Avoid stirring too violently (proteins denatureif subjected t o shea ring st resses; if you see bubbles formin g, you are dena tu ring yourproteins). St ir for a n a ddit iona l 15 minutes a f t er you f inish a dding the amm oniumsulfate (this gives the ammonium sulfate a chance to dissolve, and allows theproteins a cha nce to equilibrat e to the presence of th e am monium sulfa te).

(Ammonium s ulfat e precipita tes proteins. Different proteins precipita te a t differentconcentr a tions of a mmonium su lfa te. You a re using 60% a mm onium sulfa te; th ismeans t ha t t he amount of ammonium sulfa te you a re adding is 60%of the maximumamount of ammonium sulfate that will go into solution. Most, although not all,

proteins precipitate at 60%ammonium sulfate.)

6. Centrifugation Centr ifuge th e sa mple (a s before). Pour th e superna ta nt in to aseparate container while keeping the pellet in the centrifuge tube. Save bothsuperna ta nt a nd pellet in t he refrigerator unt il the next lab period. The LDH should bein the pellet.

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Prelab Questions #2B

1. You put a cuvette into th e spectrophotometer an d obta in a n a bsorba nce rea ding of2.4. I s th is a believa ble reading? H ow much of th e light is a ctua lly rea ching t hedetector?

Your inst ructor t a kes the cuvet te , f licks i t w ith a f inger , a nd t hen repla ces i t in t hespectr ophotometer. The rea ding is n ow 0.314. Wha t d o you t hin k ha ppened?

2. Why d oes the La b Ma nua l recommend t ha t y ou gently resuspend th e amm oniumsulfate pellet?

3. Wha t w ould h a ppen if you diluted your protein solut ion t o 20 ml a nd loaded t he

entire solution on th e Desalt ing column?

4. Why d oes LD H bin d C iba cron blue?

5. Why d o you w ish t o rea d A280 for y our C ibacron B lue column fra ctions?

6. You h a ve st ock solutions of 1 M Tris ba se, 1 M H Cl, 0.2 M 2-merca ptoetha nol, 0.1M P MS F, a nd 0.5 M ED TA. How w ould you ma ke up 200 ml of extra ction buffer?

Extraction buffer:10 mM Tris -H C l (pH 7.4), 1 mM 2-Merca ptoet ha nol, 1 mMP MSF a nd 1 mM ED TA.

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Experiment 2BPurification of Lactate Dehydrogenase from Chicken, Part II

Reagents

Tris-PMSF Buffer10 mM Tris -H Cl (pH 8.6)0.5 mM 2-Mercaptoethanol1 mM PMS F

NAD Buffer10 mM Tris -H Cl (pH 8.6)0.5 mM 2-Mercaptoethanol1 mM lithium lactat e1 mM N AD

NADH buffer

10 mM Tris -H Cl (pH 8.6)0.5 mM 2-Mercaptoethanol1 mM NADH

Econopac d esalt ing columnCiba cron B lue columnTest TubesDisposab le pipets

Procedure

High concentrations of salts (such as ammonium sulfate) often interfere with

subsequent purifica tion st eps. Removing sa lt is t herefore frequently necessar y. Thenext step is intended to separate the ammonium sulfate from the protein. Thedesalt ing column is a sma ll gel filtra tion column. G el filtra tion columns separ a te onthe ba sis of size, with lar ge molecules moving thr ough t he column more ra pidly t ha nsma ll ones. The columns y ou w ill use are set up so tha t y ou can sepa ra te th e proteinfrom t he sa l t assumingyou use th e volumes listed below. If you do not follow th eprocedure, the salt will end up mixed with the protein. (Note: gel filtrationchromatography is discussed in more detail in the introduction to experiment 3D.)

1. Resuspend ammonium sulfate pellet Add 1 m l of Tris-P MS F buffer t othe a mmonium sulfat e pellet . Gently (a void bubbling) mix th e buffer a nd t hesolid ma teria l unt il the pellet dissolves. Keep on ice as m uch a s possible duringth is procedure. Ch eck th e volume of th e mixture (it sh ould not be more th a n 3ml). If n ecessary, a dd buffer so tha t the t ota l volume is 3 ml. If it is impossibleto resuspend t he pellet in less tha n 3 ml of buffer , a sk your instr uctor i f youcan use tw o desalt ing columns.

2. Load the desalting column When y ou obta in t he column, it should cont a insome buffer a bove the frit (th e plast ic cover on t he column resin). Remove this

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buffer, a nd load 3 ml of th e mixture on t he desalt ing column. Allow the liquid t odra in to the frit. Disca rd th e flow thr ough.

3. Elute the protein from the desalting column Add 4 ml Tris-P MS F bufferto the column, a nd collect the flow th rough (th is conta ins your LD H protein, sokeep it on ice).

4. Measure and record the volume of the flow through. (Note: if you ra nmore tha n one desalting columns, combine the flow through eluents in asingle tube, an d mea sure the tota l volume.)

5. Save three 0.1 ml aliquots of th e flow t hrough eluent (label th em desalt eda mmonium sulfat e fraction).

The Cibacron Blue column is an affinity column. It specifically binds dehydrogenase-ty pe proteins (a compound structura lly similar to NADH is cova lent ly a t t a ched toth e column). You a re using th e column t o separa te th e LDH from the other proteinsin the mixtur e. Wha t you w a nt to do is a dd the mixture t o the column, and thensequentia lly remove:1) a ll proteins th a t do not bind t o th e column ,2) a ll proteins t ha t bind loosely to th e column , an d fina lly,3) th e LDH from the column.

P rote ins , NAD, a nd NADH a bsorb a t 280 nm. You can determine w hether a t leas tone of th ese ma teria ls is present in t he eluent from the Ciba cron blue column bymeasur ing the a bsorba nce of the fra ctions. Bla nk th e spectrophotometer aga inst a ir .Read th e absorban ce of a cuvette w ith Tris-P MSF buffer in it (the a bsorba nce shouldbe about 0.4). Read th e absorba nce of your Ciba cron blue column fra ct ions: i f theabsorbance is less th an ~0.1 above th e absorbance of the T r i s-PM SF bu ffer, go on to the

next step. After rea ding t he absorba nce, put th e sam ple ba ck in th e same t ube, coverthe tube w ith pa ra film, a nd st ore the tube on i