I 1

GeNel"

GeNei" lon Exchanoe

Chromatography-
TeachingKit
Manual

Cat No. New Cat No.

KT4O 106191
FevisionNo,: 00061204
lon Exchafig€ Chromarography GeNei-

CONTENTS

PageNo.

Objective 3

Principle 3

KitDescription

MatedalsProvided 7
.l Procedure I

Resuft 13

Appendix 14

Ordedng
lnfomaiion
lon Exchange Chromalography GeNei'" lon Exchange Chromalography GeNei""

Obiectives:
protern punlication by ion exchange
_.,_To_l6arn
cnromalography involvingthe fo owind expenments:
. Purilicationot LysozymeJs r9
Cl\,4_c6ltutose.
. Estrmation of Lysozyme actlvrtv
t Eslimationof proteinconcentr;lion.

Principle:
lon'exchangecl^[Link]
works on lhe basic
[Link] opposilelychargedparltcl€s
are altracledto
[Link] stationary pnaseconsistsof f,xedcharges
on a sorro,support.
Thesechargescan be eithernegativj
[Link] two lypes of ;on exchanders oi
calon and anion exchangers. i.e,,

exchangerpossessnegafi!e,ycnargedgro{,/ps
- ,Cat,on
and altractDosilivety
[Link].
eb, Ca,U-oxyfieitrii
cerdose or [Link]-n
exchanlerhaire
posrrvetychargedgroupsthal arrracrnegativety;harged
l:l:"1':: ?19 thusseparare
anronic
[Link],
uremyramlnoethvt-celtutose

Proletnsare complexamphotytesLe., they have

^-_
posrlve bolh
and negativechargesano can oe separatedfrom a
rnrxrurcolcomooundson the basisolletpositive
or regative
cn^arge_lhat they carry tsoerectricpoinl bt a protein
[pt1rs
rre pFlat.M/htc'tnelchargeis /ero (t e.,[Link]
'tS
dnonegatvechargesare equal).[Link],proteinswill
have
:Jl:r^a_l:r l'€sativechargeor net posir,vechargedepending
01 rre pH_otsotrliotandthus.it is possible to useerlhera;
anronexcnangeror a calionexchanoer

O [Link].'. 2006 @ sa.gdor9 Gfrt, 2006

lon Exchange Chromaiography GeNei"" lon ExchangeChromarography GeNei'"

In ion €xchangechromatography, a solutionconlaining Kit Oescription:

proteinof inl€reslis applied-tothe ion exchangerProlein
bindingto lhe ionexchanger is dependsnl on netchargeol Using this kii. students wi carry oul purificati; ol
.
rysozym€
the proteinat thalparticular
pH andon theionicstrength ol trom chichen 699 whilg by ion exchanoe
lhe [Link] thenelutedout homthe [Link] of tysozymeis 10.5and rl carnesa iet
slationaryphas€by increasing the concontrationot counler posrrvecnargeal pH bstowj0.5. H6nce,at pH 7.0 it
brnds
ionsorbychanging pH,whichaltersthechargeontheprotein. charged cotumn or a cation exchanger,
:..,':99!:ygly
r.M-ue u|os€ supptiedin lhe [Link] buffer (pH 9.0) witi
Weaklychargedproteinis displacediiom the stationary
phasewilh loworc$ncentration ol counl€rionslhanhighly men Deused to rcmovethe proleinlhat have pl less thanor
[Link] separalion ol proteinbased 11illt-0,-10:I"lhhg *nici, tygozym€
wrrtbeetut€d
outby
uponits net charge. Increasng lrationoI calions. Caltonscompetewitit
.concen
posilivelydhargedgroupsol tysozyrhstor bindingsites on
Extentot pu licationol a protein(e9., enzyme)can be tne coumn, resultingin lhe elulionol lysozyme.
delominedby compulingils [Link]
is the ralio of enzymeaclivityto massof proteinin the St![Link]" wilt esrimate lysozyme activiry ano prolern
-
concenrratrcnot the crud€ and purifiedsamptesas filllows:
6ample,usuallyexpressed as unitsof activityper milligram
ofprotein(U/mg). Asthe€nzymeispuritigd (lhrough a number
ol steps)otherproteinsin lh€ mixtur€are eliminated while . Enzymo Activity ol lysozymeis detemined usino the
moslof the enzymeaclivilyis retainecl, lhls resultsIn an bactenumMtcrca)ccus luleus.A suspensionot intactba;eda
increasein the specificactivityof the [Link]€,by rs_-[Link] light skongtyat 450 nm. As tysozyme
d€termining specificaclivitybeforeandafterpurification,one DreaJG doll/n,the,cerl
wa|s,.bacterial,nefibranesbreakopen
can determine the foldpu ficationandyieldof the enzyme. orie ro [Link] breakoownproduclsdtssolv€incl
rne suspensionbecomesclearer Thus. as lysozymeacts,
lhe aDsoftance ol the subslrate suspensio oecreases.
Hence, one unit o[ lysozymeis delined as the amohl ot
ty-sozymethal will produce a decreasejn aosoroanceat
450 nm of 0.001 absorbanc€units/minute.

O 6..9.10r. G...i, 2006 O B.{[Link] c.n.t, 2006

lon Exchange Chromatography GeNei"" lon ExchangeCh romatography GeNei'"

Protein concenlralion of lysozyme is eslimalod by Materials Provided:

measuringlhe UV absorbanceat 260 nm and 280 nm. This
value wrll bs Us6d-localculatesp€ciiicacUvityand yleld of The list below provid€sinlomation about lhe mateials
lysozym€. Specificaclivity ol lysozym€ before and atier suppliedin the kit. The produclsshould bo stored as
purilacation will be estimatedby comparingils aclivitywith [Link] the kit wilhin 6 monthsof arrival.
lhal ol a slandardlysozymewhos€specrficactvily is known.
Ouanlity
KTAO I fhe kit is designed ta catry oul S lysozyme
puritication expeiments by ion exchange lMalerial KT4O Store
chrcmatogEphy. (5 erpls.)
CM-oellulose 5ml 4'C
Duration ol experimeni: Approximalely5 hours
10XEquilibralion buffer 25 ml 4'C
(DH7.0)
10X Washbuffer (pH 9-0) 25 ml 4'C
5X Elutionbufler l5 ml 4"C
Neulralizingsolution 5ml 4.C
5M Sodiumchloride 10ml 4,c
o-5 M PhosphatebLrffer 25 ml 4'C
(pH7.0)
Tubefor mixinq 1 N o. 4'C
3 Nos. 4'C
LvsozvmeSlandard 2x1mg 4'C
Column 1 No. 4'C

MaterialsRequired:
Equipment; Centrifuge,
Specvophotometer.
Reagents : Chickenegg,Dislilledwater-
classware; Beakers,Testtube5.
Other Requirements: Tips,
Columnsland,Micropipette,
Ouartzcuvelte,

O [Link]. 6enei, 2o0€ @[Link],lon G.n.t 2006

 lon ExchangeChromalography GeNei* lon ErchangeChromalography GeNei"

Note: 3 . Removeth€ bottomcao and eouilibratelhe columnwith

. Read the entite ptocedute betore starting the 50 mlof 1X equilibralion
buffea
4 . Breakan egg, collectth6 egg whjt€separately.
. Handle neut@lizingsolution carcfully as it is cofiosive. 5 . To 6 ml of egg white,add an equal volum€of distill€d
. Storc the Mictococcus luteus (Mlu) vial al 4oC betorc water,Mix in lhe lube providedlor 10 minutesto get a
rcconstitution- homogenoussolution-
. Reconstitutethe Mbrococcus luteus vial with 0.1 ml of 6. Adiustthe pH ol lhe egg whiteto 7.0, by slowlyadding
0.067Mphosphate buffen pH 7.0 (PB). Store at '20"C.
Use one vial as substrate for 2 expeiments,Gently
handle the rcconstituted Mlu vial.
I t..
neutralizingsolution.
Note:Solutionwill turn slightlyturbid
C€nlrifuge the egg while solution at 6000 rpm for
. Reconstitute the lysozyme standard with 1 ml of I 10 [Link] supernalanl.
PB. Store at 4'C and use within 3 months. Use one viat L Save 0.5 ml of the supernatantlor measurementol
for 3 expeimen6. iysozymeactivity,label this as crude sample.
. Uae a Quaftz cuvette to measure absotbance at A@ Load rest of the suDernalant to eouilibrated
CNI-Cellulose column.
. Mix CM-Cellulos, column material gently to make a 10. Feplace lhe top and botlom caps of the column and
unilorm suspension, beforc packing. incubatefor t hour at room temperalurewiih intemittent
. Each packec! CM-cellulose column can be used for mrxrng,
maxinum 3 tim6s, 11. Afteranhour,[Link]
. Do not tet the column go dry. plpolteoul or decantthe supernatant withoutdisturbing
. B ng the reagenls to rcom tempenlurc beforc allluton. thg column.
. Dilute the rcquircd amount ot standad lysozyme,itst 12. Wash the column with app.oximately30 to ,lO ml ol
I X wash buffer
. For prcparation ol working concenttation of rcagents/ 13. Elute lysozymefrom lhe column using 15 ml of
bufferc, refer appendix.
t 14.
1X elutionbulret
Startcollectingthe eluat€in t€sttubesas 2 ml fractions.
Procedure:
Purification of Lysozyme using CM-cellulose: x 15.
MonilorOD al Aa and pool the lraclionsthat show
A2D0,5 and abov€.Labellhis as eluate
Wash the columnwith 10 ml of llv NaCl.R€placelhe
1. Wash lhe emptycolumnwith hot water (90"C). top and [Link] at 4'C, lor nexl use.
2. Fix th6 columnlo the [Link] top cap of the Note: The packed column can be reused two more
column and pack the column with 2.5 ml of times. Discardthe column materialafter 3 uses
CM-Cellulose. and oack with kesh 2.5 ml Clv-cellulose material.

@a€ns.ro6 G.n.t, 20{)6 @ [Link]. G.n.l, 2006

lon Exchange Chromatography GeNei" lon ExchangeChromatography GeNei'"
Estimalionol ProteinConcenlration; Estimation ol Lysozym€ activity:
Crude sample:
L [Link] crudesample20 timeswith PB (i.e.,0.2 mt 1, Dilule requiredamountof. tecorystituted standard
ol egg whitemade upto 4 ml). lysozyme1:3 Le., (0.3 ml ol standardlysozyme
+ 0.6 ml ol phosphatebullef) to bring down lhe
2. Zeto 7he speclropholometer against phosphate butfer concentrataonto 0.33 mg/ml(trom1 mg/mtto 0.93 mg/
blank. mr).
J. Measureln€ absoroanceat A.d and A-s, Note: Slore the renrainingtysozymeat 4"C and
4. Use the tollowingformulalo calculaleconcenlralionof use as slandardlor lwo more experiments.
proteinin crude sarhplei
2. Diluie the crude sample and eluted sample lo
Prol€inconcentralion
= t(1.55x A,@)- (0.77x Ad)l x dilutionlaclor I 0,33 mg/ml based on the prolein conc€ntration
estimaled. For example, if the concentrationis
tactor= 20).
(dilulion 1 mg/ml,dilute it three limes with phosphatebuiferto
mgml bing down lhe concenlralion to 0.33 rng/mt.
3. Zero the spectrophotometer againstphosphatebuffer
Eluate: blank.
1. Dilutethe €luate 10 times with PB (i.e.,0.3 ml made Dilulerequjredamountol M/uin phosphalebuftersuch
upro 3 ml). that Ad is belween0.5 and 0.7.
2. Zero the spectrophotometer againslphosphatebuffef Note: Store the remaining reconstituted M/u
blank. al -20"C and use as substrate for one more
3. Measurethe absorbanceal Are. experiment.
4. Use lhe followingtormulato calculateconc€ntrationof 5. Take 3 ml o{ diluted M/1./substralein a cuvette and
proteinsin eluate. measure lhe absorbanceal A{s against phosphate
Prolein conc€ntration [Link] is Ar$ al '0' second.
6. To lhe substrate,add 50 !i of standardlysozymeand
_ A2sox dilution,actor m9ml note the time-
2.55 7. lvlixthe contenlsof cuvettefor 15 seconds.
8. Measurelhe absorbanceexactlyafter 60 secondsof
where,dilutionfactor= 10 additionof lysozyme,
and 2.55 is the extinctioncoetficientof lysozymei.e.,
9- Repealsteps5 to 8 for the crude and ellted samples.
A@ of I mg/mlol lysozymeis 2.55.
10. Note down lhe readinosas in table 1.

@A..[Link] [Link], 2ooo 10 o s.nsaro4cene!,2006 ll

 l o n Ex c hangeChr om a to g ra p h y GeNei'^' l on E xchangeC hromatography GeNei'"

Appendix: Ordering Information

Preparationof 1X equilibration butfer and 1X wash
buifer: Djlule requiredamountof 10X bufler ten timeswith S ize Cat #
dislilledwaler.(For eg., 1 ml of bufler+ 9 ml of distillsd
ceNeirM lon Exchange 1 Pack KT40
Chromatography TeachingK(
Preparationof 1X elutionbufter:Diluterequrred amount {Consumables for 5 exPerimenls)
ol 5X bufferfive times with distilledwater.(For eg., 1 mt of
bulfer+ 4 ml of dislilledwate4.

Preparationof 1M sodium chloridesotution:Dilute5l\rl

sodiumchloridesolutionfive timeswilh distiltedwater

Preparationof 0.067Mphosphate buffer: [Link]

phosphateblffer 7.5 times with disiitledwaier to oet a final
concentrationof 0.0671V.

€ [Link] Gen€i,2006 14 O BanoalorcGen€|,20oG

lon Exchange Chromatography ceNei'" lon Exchange Chromalography GeNei'"

Time Result:
Standard Crude Eluate Calculateand rcport fold puritication
of lhe enzyme
lysozyme.
0 sec.
60 sec. c1 c3

{ln ms) Yield {U)

Tabte 1: OD readingsol lysozymeactivily. C1xC2 (U/ms) C3rC4

Estimatlonot specllicactivityot Lysozyme:

Specificactivit ol lysozyme
in crudeandelltedsamples
is calculatedby comparing theOD readingsot tho samples
with that of slandardlysozyme, whosospecificaclivilyis

Aclivy inU/ms- 44 atlhcsbpdasl:

aa& /[Link] lheslandard
-t!0i0-du9&{&eli!!V

AA@ is lhe diflerence in Ae b€tween 0 sec and 60 sec

'Activity ol standard is 48,000 U/mg.

Estimation of Fold Purification:

Fold purificalion . [Link]-sanple
Sp€cificactivityol crudesample

Thls ls a measure of efliciency of pu licalion of lysozyme

uslnO ion exchange chromatography.

Estlmation of Yield:
Yield = Proteinconcentrationof eluate x lotal volum€ of eluale
=m s

O [Link]€G6n.l,2006 Gen.i,2006
o Bansaror€ 13