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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