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Ion Exchage Chromatography

Ion Exchage Chromatography

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Published by Ravish Rana

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Published by: Ravish Rana on Jun 18, 2012
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03/03/2015

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Objectives:To perform protein purification by ion exchange chromatography .Principle :Ion exchange chromatography works on the basic principle that oppositely chargedparticles are attracted to each other .The stationary phase consists of fixed chargeson a solid support .These charges can be either positive or negative .Hence , there aretwo types of ion exchangers i.e. cation and anion exchangersCation exchanger possess negatively charged groups and attract positively chargedmolecules , e.g. Carboxymethyl – cellulose or CM cellulose .Conversely , anionexchanger have positively charged groups that attract negatively chargedmolecules .e.g. , Diethylaminoethyl – cellulose .Proteins are complex ampholytes i.e. they have both positive and negative chargesand can be separated from a mixture of compounds on the basis of net positive andnegative charge that they can carry .Iso -electric point of a protein (pI) is that pH atwhich its net charge is zero (i.e. the number of positive and negative charges areequal ) . Therefore , Proteins will have either a net positive or negative chargedepending on the pH of the solution and thus , it is possible to use either an anionexchanger or a cation exchanger .In ion exchange chromatography , a solution containing protein of interest is appliedto the ion exchanger .Protein binding to the ion exchanger depends on the net chargeof that protein at that particular pH and on the ionic strength of the mobile phase.Bound protein is then eluted out from the stationary phase by increasing theconcentration of counter ions or by changing pH , which alters the charge on thatprotein .Weakly charged ions are displaced from the stationary phase with lowerconcentration of counter ions than highly charged proteins .This results in theseparation of the protein based on its net charge .Extent of purification of a protein ( e.g. ; enzyme ) can be determined bycomputing its specific activity .Specific activity is the ratio of enzyme activity tomass of protein in the sample , usually expressed as units of activity per milligramof protein ( U/mg). As the enzyme is purified ( through a number of steps ) otherproteins in the mixture are eliminated while most of the enzyme activity is retained ,this results in an increase in the specific activity of the enzyme .Hence , bydetermining specific activity before and after purification , one can determine thefold purification and yield of the enzyme .
 
The extraction and purification of lysozyme will be carried from chicken egg whiteby ion exchange chromatography . The pI of lysozyme is 10.5 and it carries a netpositive charge at the pH below 10.5 . Hence , at pH 7.0 , it binds to negativelycharged column or a cation exchanger .Wash buffer ( pH 9.0 ) will then be used toremove the protein that have pI less than or equals to 9.0 .Following which ,
 
Lysozyme will be eluted out by increasing concentration of cations . Cationscompete with positively charged groups of lysozyme for bindibg sites on the column ,resulting in elution of lysozymeLysozyme activity and protein concentration of the crude and purified sample are asfollows ;Enzyme activity of the lysozyme is determined using the bacterium
 Micrococcusluteus .
A suspension of intact bacteria
is cloudy ,absorbing light strongly at 405 nm
.As lysozyme breaks down the cell walls , bacterial membranes break open due toosmotic shock , the breakdown product dissolve and the suspension becomesclearer .Thus as lysozyme acts , the absorbance of the substrate suspension decreases.Hence , one unit of lysozyme is defined as the amount of lysozyme that will produce adecrease in absorbance at 450 nm of 0.001 absorbance units/minute .Protein concentration of lysozyme is estimated by measuring the UV absorbance at260 nm and 280 nm This value will be used to calculate specific activity and yield of lysozyme .Specific activity of lysozyme before and after purification will be estimatedby comparing its activity with that of a standard lysozyme whose specific activity isknown .Materials required
:CM –cellulose10 X Equilibration buffer ( pH 7.0)10X wash buffer ( pH 9.0 )5X Elution buffer  Neutralizing solution5M Sodium chloride0.5 M phosphate buffer (pH – 7.0 )Tube for mixingMicrococcus luteusLysozyme standardColumn
Equipment required
Centrifuge , Spectrophotometer 
.Reagents:
Chicken eggDistilled water 
 
Glasswares
Beakers , test tubes
.Other requirements : 
Column stand , micropipette , Tips , quartz cuvette.Procedure : 
Purification of lysozyme using CM cellulose ; 
1.Wash the empty column with hot water ( 90 degree Celsius )2.Fix the column to the stand .Remove the top cap of the column and pack thecolumn with 2.5 ml of CM –cellulose .3.Remove the bottom cap and equilibrate the column with 50 ml of 1X equilibration buffer .4.Break an egg and collect the egg white separately .5.To 6 ml of egg white , add an equal volume of distilled water .Mix in the tube for 10 min to get the homogenous solution .6.Adjust the pH of the egg white to 7.0 , by slowly adding neutralizing solution .7.Centrifuge the egg white solution at 6000 rpm for 10 minutes .Collect thesupernatant .8.Save 0.5 ml of the supernatant for measurement of lysozyme activity ,label thisas crude sample .9.Load rest of the supernatant to equilibrated CM cellulose column .10.Replace the top and bottom caps of the column and incubate for 1 hour at roomtemperature with intermittent mixing .11.After an hour , allow the column material to settle .Slowly pipette out or decantthe supernatant without disturbing the column .12. Wash the column with approximately 30 – 40 ml of 1X Wash buffer .13.Elute lysozyme from the column using 15 ml of 1X elution buffer .14.Start collecting the eluate in test tubes a 2 ml fractions .Monitor OD at A 280and pool the fractions that show A 280 0.5 and above .Label this as eluate .15.Wash the column with 10 ml NaCl .Replace the top and bottom caps .Store at 4degree Celsius , for next use .
Estimation of protein concentration
:Crude sample:1.Dilute the crude sample 20 times with PB .( i.e. 0.2 ml of egg white made upto 4 ml .).2. Zero the spectrophotometer against phosphate buffer blank .

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