8/9/2019 i e chgraphy

1/9

I 1GeNel"

GeNei" lon ExchanoeChromatographyTeaching itManual

Cat No. NewCat No.KT4O 106191

FevisionNo,:00061204

8/9/2019 i e chgraphy

2/9

lonExchafi Chromarography GeNei-CONTENTS

.l

ObjectivePrincipleKitDescriptionMatedalsProvidedProcedureResuftAppendixOrdedngnfomaiion

PageNo.33

7I

1314

8/9/2019 i e chgraphy

3/9

lon ExchangeChromalography GeNei'"

O tangrror.Gn.'. 2006 @ sa.gdor9 Gfrt, 2006

lon ExchangeChromalography GeNei""Obiectives:_.,_To l6arnprotern punl icat ion by ion exchangecnromalographyinvolving he fo owindexpenments:. Purilication t Lysozyme s r9 Cl\,4_c6ltutose.. Estrmat ionf Lysozyme ct lvr tvt Eslimation f proteinconcentr;lion.Principle:

lon'exchangecl^romatog.aDhyorkson lhe basicp.rncrprehat opposilely hargedparltclsare altracled oeacnotner. he stat ionarynase onsists f f ,xed hargeson a sorro,support.hesecharges an be eithernegativjoiposrlve.Hence. hereare two ypesof ;onexchanders.e,,calon and anionexchangers.- ,Cat,onexchangerpossessnegafi!e,ycnargedgro{,/psandaltractDosilivetyhargea norec.rres.eb,Ca,U-oxyfieitriicerdoseor cM-cellulose. onversery.nro-nxchanlerhaireposrrvety harged roups halarrracr egat ivety;hargedl : l :"1':: ?19 hussepararenronicotecutes.s,uremyramlnoethvt-celtutose

^-_ Proletnsare complexamphotytes e., hey have bolhposrlveand negative hargesanocan oe separatedromarnrxrurc lcomoounds n thebasisolletpositive or regativecn^arge_lhatheycarry tsoerectric oinlbt a protein[pt1 srrepFl t .M/htc ' t' tS elcharges ero te. ,nLmbe.olposi t ivednonegatvecha gesareequal). he.elore, roteins il lhave:Jl:r^a_l:r l'sative harge r netposir,ve ha gedepending01rre pH_ototr l iotand hus. t s possibleo useer lher ;anronexcnanger r a calionexchanoer

8/9/2019 i e chgraphy

4/9

lon Exchange Chromaiography GeNei""In ion xchangehromatography,solution onlainingprotein f inlresls applied-tohe on exchanger roleinbindingo lhe onexchangers dependsnln netcharge ltheprotein t thalparticularHandon he onic trength llhe mobile hase. oundDroleins heneluted uthom he

slationaryhas y ncreasingheconcontrationtcounlerions rbychangingH,which ltershe harge n heprotein.Weakly hargedproteins displacediom the stationaryphasewilh owor $ncentrationl counlrons hanhighlycharged rotein. his esultsn separalionl protein asedupon tsnetcharge.Extentot pu licationol a protein e9.,enzyme) anbedelomined by compulingls specificaclivity.Specific cliviiyis the ralioof enzymeaclivity o massof proteinn the6ample, sually xpresseds unitsof activity ermilligram

ofproteinU/mg).s henzymespuritigdlhroughnumberol steps) therproteinsn h mixtur re eliminated hilemoslof the enzyme clivily s retainecl,hls resultsn anincreasen the specific ctivity f theenzyme.Henc, ydterminingpecific clivity efore ndafter urification,necandeterminehe oldpu fication ndyield f theenzyme.

O 6..9.10r. G...i, 2006

lonExchange hromarography GeNei'"Kit Oescription:. Using this kii. studentswi carry oul purificati; olrysozym rom chichen 699 whi lg by ion exchanoeclromqrographv. t of tysozymes 10.5and rl carnesa ietposrrve nargeal pH bstow 0.5. H6nce,at pH 7.0 t brnds:.. , ' :99!:ygly chargedcotumnor a cation exchanger,r.M-ue u|ossupptied n lhe krr.Washbuffer (pH 9.0) witimenDeused o rcmove he proleinhathavepl less hanor11illt-0,-10:I"lhhg*nici, tygozymrrt eetutdutbyIncreasng.concenrationoI calions.Caltonscompetewititposilively hargedgroupsol tysozyrhsor bindingsites ontne coumn, resultingn lhe elulionol lysozyme.

- St!j9.lt" wilt esrimate ysozymeactiviryano prolernconcenrratrcnt the crudand purified amptes s filllows:. EnzymoActivity ol lysozymes detemined usino hebactenumMtcrca)ccus uleus.A suspensionot intactba;edars_-croudy.lsorbing ightskongtyat 450 nm.As tysozymeDreaJGoll/n,the,cerla|s,.bacterial,nefi ranesbreakopenorie o osmotic hock. hebreakoown roducls tssolvnclrne suspension ecomes learer Thus.as lysozymeacts,lhe aDsoftanceol the subslratesuspensio oecreases.Hence,one unit o[ lysozyme s delinedas the amohl otty-sozymehal will producea decrease n aosoroanceat450 nm of 0.001absorbanc nits/minute.

O B.{g.loE c.n.t, 2006

8/9/2019 i e chgraphy

5/9

lon Exchange Chromatog aphy GeNei""Protein concenlral i on of lysozyme s esli malod bymeasuringhe UV absorbance t 260 nm and 280 nm. Thisvaluewrll bs Us6d-lo alculate pciiicacUvity nd yleldoflysozym.Specificaclivity ol lysozym before and atierpurilacation ill be estimated y comparing ls aclivitywithlhal ol a slandardysozyme hosspecrfic ctvily s known.

KTAO I fhe kit is designed ta catry oul S lysozymepuritication expeiments by ion exchangechrcmatogEphy.Duration ol experimeni: Approximalely hours

O Bang.br. 6enei, 2o0 @A.ns,lon G.n.t 2006

lon ExchangeCh oma ography GeNei'"Mater ia ls Provided:

The list belowprovidsnlomationabout he mateialssuppl ied n the ki t . The producls houldbo storedassuggested. se the kit wilhin6 monthsof arrival.

lMalerialOuanlity

StoreT4O(5erpls.)CM-oellulose 5ml 4'C10XEquilibralionuffer(DH7.0) 25 ml 4'C10X Washbuffer(pH9-0) 25 ml 4'C5X Elutionbufler l5 ml 4"CNeulralizingolution 5ml 4.C5M Sodiumchloride 10ml 4,co-5 M Phosphate Lrffer(pH7.0) 25 ml 4'CTube or mixinq 1 No. 4'C

3 Nos. 4'CLvsozvme landard 2x1mg 4'CColumn 1 No. 4'CMaterialsRequired:EquipmentCentrifuge,pecvophotometer.Reagents : Chicken gg,Dislilled ater-classware; Beakers, est ube5.Other Requirements: olumn land,Micropipette,ips,Ouartz uvelte,

8/9/2019 i e chgraphy

6/9

lon ExchangeChromalography GeNei* lon ErchangeChromalography GeNei"Note:. Read the entite ptocedute betore starting the

. Handleneut@lizingsolutioncarcfully as it is cofiosive.

. Storc the Mictococcus luteus (Mlu) vial al 4oC betorcrcconstitution-. Reconstitute he Mbrococcus luteus vial with 0.1 ml of0.067MphosphatebuffenpH 7.0 (PB). Store at '20"C.Use one vial as substrate or 2 expeiments,Gentlyhandle the rcconstituted Mlu vial.. Reconstitute the lysozyme standard with 1 ml ofPB. Store at 4'C and use within3 months.Use one viatfor 3 expeimen6.. Uae a Quaftz cuvette to measure absotbance at A@. Mix CM-Cellulos, column material gently to make aunilorm suspension,beforc packing.. Each packec! CM-cellulose column can be used formaxinum 3 tim6s,. Do not tet the column go dry.. B ng the reagenls to rcom tempenlurc beforc allluton.. Dilute the rcquircd amount ot standad lysozyme, tst. For prcparation ol working concenttation of rcagents/

bufferc, refer appendix.Procedure:Purification of Lysozyme using CM-cellulose:1. Wash he emptycolumnwith hot water 90"C).2. Fix h6 column o the stand.Remove he opcap of thecolumn and pack the column with 2.5 ml ofCM-Cellulose.

Remove hbottom aoand eouilibratehe columnwith50 mlof 1X equilibralionuffeaBreakan egg,collect h6 egg whjtseparately.To 6 ml of egg white,add an equalvolumof distilldwater,Mix n lhe ube providedor 10 minutes o get ahomogenous olution-Adiust he pH ol lhe egg white o 7.0,by slowlyaddingneutralizing olution.Note:Solut ionwi l l turn s l ight ly urbidCnlrifuge the egg while solution at 6000 rpm for10 minules.Collecl he supernalanl.Save 0.5 ml of the supernatantor measurement liysozymeactivity, abel his as crude sample.Load rest of the suDernalant o eoui l ibratedCNI-Celluloseolumn.Feplace lhe top and botlom caps of the column andincubate or t hour at roomtemperalurewiih intemittentmrxrng,Afteranhour,allowthe olumnmaterialto ettle.Slowlyplpolteoul or decant he supernatant ithout isturbingthg column.Wash the columnwith app.oximately 0 to ,lOml olI X wash bufferElute ysozyme rom lhe column using 15 ml of1X elution ulretStart ollectingheeluat n tst ubesas 2 ml ractions.MonilorOD al Aa and pool the lraclions hat showA2D0,5 and abov.Label his as eluateWash he columnwith 10 ml of llv NaCl.Rplace hetop and botlomoaps.Slorcat 4'C, lor nexl use.Note: The packedcolumn can be reused wo moret imes. Discard he column materialaf ter 3 usesand oack with kesh 2.5 ml Clv-cel lu lose ater ia l .

3.4.5.

II t..L

6.

10.

tx 15.14.11.12.13.

@ans.ro6 G.n.t, 20{)6 @Bang.lor. G.n.l, 2006

8/9/2019 i e chgraphy

7/9

lon ExchangeChromatograph GeNei"Est imal ion l ProteinConcenlrat ion;Crude sample:L Dilr.itehe crudesample20 timeswith PB (i.e.,0.2 mtol egg whitemadeupto 4 ml).2. Zeto 7hespeclropholometeragainst phosphatebutferblank.J. Measuren absoroance t A.d andA-s,4. Use the tollowingormula o calculale oncenlralion fprotein n crudesarhpleiProlinconcentralion= t(1.55 A,@) (0.77x Ad)l x dilution aclor

lon ExchangeChromatography GeNei'"

(dilulionactor 20).mgml

Eluate:1. Dilute he luate 10 times with PB (i.e.,0.3 ml madeupro3 ml).2. Zero the spectrophotometergainslphosphate uffefblank.3. Measure he absorbance l Are.4. Use he followingormula o calculate oncntrationfproteins n eluate.Prolein concntration_ A2so dilution actor2.55where,dilution actor= 10and 2.55 s the extinction oetficient f lysozyme.e.,A@ of I mg/mlol lysozymes 2.55.

Estimationol Lysozymactivity:1, Di lu le requiredamountof. ecoryst i tutedtandardlysozyme1:3 Le., (0.3 ml ol standard ysozyme+ 0.6 ml ol phosphate ul lef) to br ing down heconcentrataono 0.33 mg/ml trom1 mg/mt o 0.93 mg/mr).Note: Slore the renraining ysozymeat 4"C anduse as slandard or lwo more exper iments.2. Di lu ie the crude sample and eluted sample o0,33 mg/ml based on the prolein concntrat ionest imaled. For example, f the concentrat ion s1 mg/ml,dilute t three imes with phosphate uifer obing down lhe concenlralion to 0.33 rng/mt.3.

5.

6.

Zero the spectrophotometergainstphosphate ufferblank.Dilule equjred mount l M/u n phosphale ufter uchthat Ad is belween0.5 and 0.7.Note: Store the remaining reconst i tuted M/ual -20"C and use as substrate for one moreexper iment.Take3 ml o{ diluted M/1./ubstrale n a cuvetteandmeasure he absorbanceal A{s againstphosphatebuderblanh. his is Ar$ al '0 ' second.To lhe substrate, dd 50 !i of standard ysozyme ndnote he time-

I

m9ml 7. lvlix he contenlsof cuvette or 15 seconds.8. Measure he absorbance xactlyafter60 secondsofadditionof lysozyme,9- Repealsteps5 to 8 for thecrudeand ellted samples.10. Notedown he readinos s in table 1.

ll@A..g.lod G.mr, 2ooo 10 o s.nsaro4cene!, 006

8/9/2019 i e chgraphy

8/9

lon Exchange hromatography GeNei'^'Appendix:Preparat ionof 1X equil ibrat ionbutfer and 1X washbuifer: Djlule equired mountof 10X bufler en timeswithdisl i l led aler. Foreg., 1 ml of bufler+ 9 ml of dist i l lsdPreparation f 1X elut ionbufter:Di lute equrredmountol 5X buffer ive times withdistilledwater. Foreg., 1 mt ofbulfer+ 4 ml of dislilledwate4.Preparation f 1M sodium chloridesotut ion:Dilute l \ r lsodiumchloridesolution ive timeswilh distiltedwater

lon Exchange hromatography GeNei'"Ordering nformation

S ze Cat#ceNeirM lon ExchangeChromatographyeaching ({Consumablesor 5 exPerimenls)

1 Pack KT40

Preparation f 0.067Mphosphatebuffer:Dilute0.stvlphosphate lffer 7.5 times withdisiitledwaier o oet a finalconcentration f 0.0671V.

Bans.rorc Geni,2006 14 O BanoalorcGen|,20oG

8/9/2019 i e chgraphy

9/9

lon Exchange Chromatography ceNei'"Time

Standard Crude Eluate0 sec.60 sec.

Tabte 1: OD readingsol lysozyme ctivily.Estimatlon t specllicactivityot Lysozyme:Specificctivit ol ysozymencrude ndelltedsamplesis calculatedy comparingheOD readingst tho sampleswith hat of slandardysozyme, hosospecific clivily sAclivy nU/ms- 44 -t!0i0-du9&{&eli!!V atlhcsbpdasl:aa& /mio. l heslandardAA@ s lhe diflerence in Ae btween 0 sec and 60 sec'Activity ol standard is 48,000 U/mg.Estimation of Fold Purification:Fold purif calion . Speqifieiqliyill.qlellled-sanpleSpcific ctivity l crudesampleThls ls a measure of efliciency of pu licalion of lysozymeuslnO on exchange chromatography.Estlmationof Yield:Yield= Proteinconcentrationof eluate x lotal volum of eluale=ms

lon ExchangeChromalography

Result:Calculate nd rcport old pur i t icat ionf lhe enzymelysozyme.

o Bansaroren.i,2006

GeNei'"

c1 c3{ ln ms)C1xC2 (U/ms) Yield U)C3rC4

O B.ngllorG6n.l,2006 13