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O*+ec%i,e-
"! /o iolate an0 partially purify Immunoglobin G (IgG) from 1hole
bloo0 erum of chicken!
2! /o un0ertan0 the ariou techniue in the proce of bloo0
erum preparation!3! /o compare the protein concentration an0 purity of cru0e erum
cru0e albumin an0 immunoglobulin G (IgG)!
In%ro)!c%ion-
In thi e5periment chicken erum 1a ue0 for preparation of
immunoglobulin G (IgG)! Serum i the clear liui0 that can be
eparate0 from clotte0 bloo0! Serum 0i6er from plama the liui0
portion of normal unclotte0 bloo0 containing the re0 bloo0 cell1hite bloo0 cell an0 platelet! It i the clot that make the
0i6erence bet1een erum an0 plama! Since erum contain t1o
ma7or type of protein 1hich are globulin an0 albumin! /hee t1o
ma7or type of protein are goo0 ource of immunoglobulin!
pecially erum IgG a it i more abun0ant than Ig- IgA Ig8 or
Ig! /hree puri9cation techniue are ue0 to iolate Ig G from
erum uch a alt precipitation anion e5change chromatography
an0 0ialyi!
In thi e5periment o0ium ulfate precipitation an0 0ialyi
1ere ue0 to iolate IgG from chicken erum! So0ium ulfate
precipitation i the common ue0 techniue for the preparation of
immunoglobulin from the 1hole chicken erum! "!,g o0ium
ulphate 1a a00e0 to the erum! Since the alt concentration of
the me0ium 1a increae0 there 1a an interference 1ith the
interaction of 1ater molecule 1ith charge0 polar group on protein
molecule! /he protein molecule became le hy0rophilic
permitting greater hy0rophobic interaction bet1een protein
molecule! entually the protein became inoluble! /he altconcentration 0i6er at 1hich each protein precipitate 1ith
oerlap bet1een imilar protein! *hen preparing the cru0e
immunoglobulin fraction un1ante0 erum protein taye0 in
olution 1hile immunoglobulin 1ere precipitate0 out! /he protein
concentration in the me0ium inuence0 the precipitation limit of
the protein an0 a6ecte0 the coprecipitation limit of the protein!
;ence 1hen precipitating erum o0ium ulfate 1a a00e0 lo1ly
to aoi0 high local concentration 1hich 1oul0 0ecreae the
peci9city of the precipitation!
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8ialyi tubing i alo kno1n a <iking tubing! It i a type of
emi=permeable membrane tubing ue0 in eparation techniue
that facilitate the remoal or e5change of mall molecule from
macromolecule in olution bae0 on 0i6erential 0i6uion! In thi
e5periment 0ialyi tubing i ue0 to clean up the un1ante0 erumprotein in the comple5 biological ample uch a erum! 8ialyi
eparate protein bae0 on the i>e of the 0ialyi pore! ?rom
0iagram " the erum 1a put ini0e the 0ialyi tubing an0 tie0 a
knot at one en0! It immere0 in phophate bu6er aline! /he large
IgG molecule coul0 not pa through the 0ialyi pore o it taye0
remain in the 0ialyi tubing! /he mall protein molecule coul0
pa through the 0ialyi pore o it 0i6ue0 out into phophate
bu6er aline! After one 0ay only pure IgG taye0 remain in the
0ialyi tubing!
(a) Before 0ialyi (b) 8uring
euilibrium
8iagram ": /he olution before an0 after 0ialyi!
Me%ho)olo.'-
Par% I- Preara%ion of *loo) "er!&/ /he freh bloo0 1a allo1e0 to tan0 at room temperature for "
hour! /he bloo0 1a tore0 oernight at 4o' to allo1 clot formation!
/he erum 1a poure0 o6 an0 the ma7ority of clot 1a remaine0 in
bottom of ak! It 1a then centrifuge0 at 3%%% rpm for "% minute!
/he upernantant 1a tranferre0 to ne1 centrifuge tube an0 the
centrifugation 1a repeate0!
Par% II- Cr!)e I.G reara%ion/
4& ml of chicken erum 1a preheate0 at &@o' for 2% minute to
remoe complement protein! /he erum 1a centrifuge0 at .%%%rpm for 2% minute at 4o' to remoe upernatant an0 then the
Serum
Dialysis Membrane
Phosphate Buffer
Saline
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erum 1a 9ltere0! "!,g of o0ium ulphate (", 1) 1a a00e0
mi5e0 1irle0 an0 0iole0! It 1a then centrifuge0 at .%%% rpm
for "%="& minute at 2&o'! /he pellet 1a 0iole0 in & ml 0itille0
1ater an0 thu became cru0e IgG! /he 0ialyi tubing 1a 1et 1ith
0itille0 1ater! A knot 1a tie0 at one en0 the & ml cru0e IgG 1alea0 in an0 then a knot 1a tie0 at oppoite en0! /he 0ialyi tubing
1a place0 in "=2 liter of phophate PBS bu6er an0 left at 4o'
oernight! /he uantitatie an0 ualitatie meaurement of cru0e
erum cru0e albumin an0 partially puri9e0 IgG 1ere 0one by uing
Cano0rop pectrophotometer! 8itille0 1ater 1a ue0 a a blank!
/he aborption pectrum 1ere printe0 out an0 analy>e0!
Re"!l%"-
0/ A*"or%ion "ec%r!& of cr!)e "er!&
1/ A*"or%ion "ec%r!& of cr!)e al*!&in
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2/ A*"or%ion "ec%r!& of ar%iall' !ri3e) i&&!no.lo*!lin
G (I.G#
Re"!l% Anal'"i"-
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Sa&le
Pro%ein
concen%ra%ion
(&.4&l#
A156
(n&#
A1764A15
6
Cr!)e "er!& 3"!".4 3"!".4 %!@&
Cr!)e al*!&in 2,!,%" 2,!,%" %!,.I&&!no.lo*!lin G
(I.G#2!@,4 2!@,4 %!&,
Di"c!""ion-
8ialyi 1ork by 0i6uion 1hich i a proce reult from the
ran0om moement of molecule in olution an0 caue the net
moement from area of higher to lo1er concentration untileuilibrium i reache0! In thi proce un1ante0 molecule ini0e a
ample=chamber 0i6ue through a emi=permeable membrane into
another chamber of liui0! /he large molecule cannot pa through
the pore of membrane thu they 1ill remain in the ample
chamber! $n the other han0 the mall molecule can 0i6ue acro
the membrane an0 thu reach euilibrium acro the 1hole olutionD
therefore the concentration of thoe mall molecule 1ithin the
ample i re0uce0!
Aborbance at 2,% nm (A2,%) i ue0 to 0etermine the protein
concentration bae0 on the aborbance of +< light by the aromatic
amino aci0 tryptophan an0 tyroine an0 by cytine 0iul90e
bon0e0 cyteine rei0ue in protein olution! ?rom the reult 1e
obtaine0 the cru0e protein ho1 higher protein concentration than
IgG! Before 0ialyi there are a lot of impuritie ini0e the cru0e
protein epecially alt an0 other contaminant! After 0ialyi 1a
0one ome un0eire0 contaminant inclu0ing the alt 1ere
remoe0 thu the IgG contain le contaminant! /he protein
concentration alo 0ecreae becaue ome tiny protein 1hich
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maller than the pore of 0ialyi tubing hae been remoe0! /hi
can be een from the A2@%A2,% ratio! In general pure protein ha
an A2@%A2,% ratio of 2!%! /he cru0e protein houl0 be ha a higher
impurity than IgG but our reult ho1 a 0i6erent reult! It may 0ue
to ome e5perimental error!
Albumin preent more than half of all bloo0 plama protein!
/hey are rea0ily olubili>e in 1ater! /he ", 1 of o0ium ulphate
1a a00e0 to olubili>e the albumin in the olution an0 become
upernatant after centrifugation! In a00ition o0ium ulphate alo
allo1 the alpha=globulin (IgA) to 0iole in olution 1hile the
other protein inclu0e0 gamma=globulin (IgG) 1a precipitate0 out!
In thi 1ay IgG can be fractionate0 from each other by uing the
olubility of protein!
8ialyi membrane ha 0i6erent thickne an0 pore i>e!
/hicker membrane i tougher but it 1ill retrict olute o1 an0 thu
euilibrium i reache0 more lo1ly! IgG i the pre0ominant
immunoglobulin of internal component uch a bloo0 an0
cerebropinal ui0 1hich i aroun0 ,% of the total
immunuglobulin! It i alo the mallet immunoglobulin 1hich ha
a molecular 1eight of "&%%%% 8alton! /herefore it can rea0ily
0i6ue out of the bo0yE circulation into the tiue! /he pore of the
tubing mut be maller than the i>e of IgG in or0er to hol0 it ini0e
the tubing! In contrat Ig- i a macroglobulin 1hich i the larget
immunoglobulin 1hich ha a molecular 1eight of .%%%%% 8alton!
In other 1or0 IgG can be hol0 in the tubing a 1ell a other
immunoglobulin 1hich are larger than IgG! A a reult the 0ialyate
i till containing other clae of immunoglobulinD therefore further
puri9cation of IgG i nee0e0!
Bloo0 erum i bloo0 plama 1ithout 9brinogen or the other
clotting factor! It i clearer than plama becaue of fe1er protein!
8uring the preparation of bloo0 erum in thi e5periment the bloo01a tore0 oernight to allo1 clot formation a the 0iagram belo1!
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8iagram 2: Serum an0 the clotte0 bloo0!
/here are ome protocol 0uring the preparation of bloo0
erum in thi e5periment! /he bloo0 1a tore0 at roomtemperature 1hich hiel0e0 from light! /he cell houl0 not be
refrigerate0! /he upernatant 1a tranferre0 to centrifuge tube
carefully to aoi0 the 0iruption of cell layer or tranfer any cell!
Some precaution mut be carrie0 out throughout the e5periment to
obtain a more accurate reult! A clean pipette 1a ue0 an0 gloe
1ere 1orn to prepare bloo0 erum to aoi0 contamination! /he
0ialyi tubing 1a clippe0 1ell on both en0 to enure that there i
no olution eepe0 through an0 thereby a6ecting the reult!
Concl!"ion-
8ialyi i one of the puri9cation techniue to iolate
immunoglobulin IgG from erum! 8ialyi remoe un1ante0
contaminant an0 thu the partially puri9e0 IgG contain le
contaminant an0 lo1er protein concentration! All the ample are
not pure 1hich hae lo1 alue of A2@%A2,% thi may 0ue to ome
e5perimental error an0 therefore precaution are nee0e0 to obtain
a pure ample!
Reference"-
8ialyi -etho0 for Protein Feearch /hermo ?iher Scienti9c
2%"&! Fetriee0 "&th Coember 2%"& from:
http:111!thermo9her!commyenhomelife=cienceprotein=biologyprotein=biology=learning=centerprotein=biology=reource=
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librarypierce=protein=metho00ialyi=metho0=protein=
reearch!html
Immunoglobulin 5plaine0 by 8r! Guy Sher1oo0! Fetriee0 "&th
Coember 2%"& from:
http:111!i1mf!comite0efault9le0ocImmunoglobulin5plai
ne0!p0f
/he ue of o0ium ulfate a the globulin precipitant in the
0etermination of protein in bloo0 by Paul ! ;o1e ! Biol! 'hem!
".2" 4.:.3="%#! Fetriee0 "&th Coember 2%"& from:
http:111!7bc!orgcontent4.".3!full!p0f
8ialyi in Protein Feearch: +n0ertan0ing the Baic pote0 by
Protein -an on 2,th -ay 2%"4! Fetriee0 "4th 8ecember 2%"& from:
http:info!gbiocience!comblogbi0".#&&&8ialyi=in=Protein=
Feearch=+n0ertan0ing=the=Baic
Protocol for the Preparation of Bloo0 Plama an0 Serum
ProImmune Himite0 2%%.! Fetriee0 "4th 8ecember 2%"& from:
http:111!proimmune!comecommercep0f9lePF3"!p0f