Class Note for BIOC 463A at UA
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Date Created: 02/06/15
BIOC 463A Expt 4 Column Chromatographic Methods Column Chromatography Chromatography is the process use to separate molecules based on SOME physical property of the molecule 0 Mass ie size 0 Charge 0 Affinity for ligands or substrates 0 Hydrophobic interactions Two phases in EVERY chromatography experiment Stationary phase a surface or resin that is inert 0 Mobile phase Comprised of the solvent and the sample eluant Introduction of the mobile phase can either be done by a gravity feed siphoning system or by a pumping device usually peristaltic In most of the figures for this chapter see below gravity feed systems are used Seperation occurs due to VARYING degrees of interaction of sample with the stationary phase BIOC 463A Expt 4 Column Chromatographic Methods Interaction of sample with stationary phase can be modulated by changing the solvent conditions ie pH ionic strength competitive ligands etc For column chromatography stationary phase is referred to as resin or gel or matrix Three primary types of RESINS 0 Gel Filtration Size Exclusion SEC Molecular Sieve 0 Ion Exchange IEC o Affinity ligands substrates or tags alocasaA Ext16 Cnlumn chmmmnqmn m Melhnds pmuju mum mmnx mum Mum BIOC 463A Expt 4 Column Chromatographic Methods Size Exclusion Chromatoqraphv Uses 1 Separation and purification of proteins 2 Determination of Molecular Weight 3 Desalting ie removing small molecule salts protein samples 4 Change the pH and ionic strength of the buffer that the protein is in Molecules separated according to their Stokes Radii Assume that the Mass of protein is proportional to its Volume For spherical globular proteins radius of protein hydration sphere is proportional to molecular mass or weight BIDC 4A Expt 4 Column Chromatographic Methods Suppose you have a spherical protein with a Stokes radius of 5 nm 50 A Then v 43nr3 524 nm3 EIDC 463A Expt 4 Column Chromatographic Methods For non spherical proteins the radius of the sphere described by the rotation of the molecule about its longest axis What would be the Stokes radius of a rod shaped protein that has a cylindrical radius of 15 nm V 524 nm3 rrrzh 111 5 nm2h gt s h m Stokes radius 37 nm For protein of same Volume the SR of rod 7 x SR sphere Both proteins have the same Volume or Mass but the rodshaped protein will appear to have a much larger size due to larger Stokes radius as it tumbles through a gel filtration column How does this influence interaction with SEC resins BIDC 4A Expt 4 Column Chromatographic Methods SEC resins are hollow beads prepared by cross linking a polymer such as Dextran or Acrylamide The beads have a Wifer Balllike structure with more or less discrete hole or pore sizes 625 G100 The pore size is determined by the cross linkerlpolymer ratio High ratio small pore size low molecular weight cutoffs Low ratio large pore size high molecular weight cutoffs Crosslinker figure BIOC 463A Expt 4 Column Chromatographic Methods Crosslinking results in a limited range of pore sizes rather than a single precise size This allows for a molecular weight range over which the resin is effective see Table 41 below A word of caution the larger the pore size less crosslinking the more collapsible the resin Crosslinking adds structural integrity especially for the dextran based resins i e Sephadex Under high pressure the higher M WT cutoff resins can collapse because of their LESSER degree of crosslinking creating an almost impermeable barrier at the bottom of a column Polyacrylamide ie the BioRad P series resins are significantly less prone to collapse and are often preferred for these applications BIOC 463A Expt 4 Column Chromatographic Methods gtlngqgl chipmangi phyquotM oid V5 xi 1 GEL PRODUCT EXCLUSION SIZE MW RANGE MATERIAL SOURCE AISM 15 X 106 8 x 104 12 x 105 Agarose Bioer A5M 5 x 10 4 X 104 4 X 105 Agarose Biomd A05M 5 x 105 1 x 10445 x 104 Agarose Biorad Sepharose 4B 2 x 107 8 x 103412 x 100 Dextrun LKB Sepharose 6B l X 107 4 x 10H x 106 Dextran LKB Sephndex G200 5 X 105 4 X 10442 x 105 Dexlrun LKB Sephadex 6150 2 X 105 2 X 104712 X 104 Dextran LKB Sephadex G100 1 x 05 8 x 10378 x 104 Dexmm LKB Sephadex G75 0 X 104 4 x 1034 X 104 Dextran LKB Sephadex G50 4 X 104 2 x 1013 x 104 Dexlran LKB Sephadex G25 3 x 104 08 X 1034 x 104 Dexn39an LKB Sephadex G 10 8 X 103 04 x 1034 X 103 Dextran LKB PIOO l x 105 8 x I03 8 x 104 Acrylamide BioRad P60 6 x 104 3 x 103 1 x 104 Acrylamide BioRad P30 4 x 104 2 x 103 2 X 104 Acrylumide BioRad P10 2 X 104 2 x 10341 x 04 Acrylumidc BioRad P6 a X 103 08 X 1034 X 103 Acrylamidc BioRad P2 8 x 103 1 x 10242 x 103 Acrylumide BioRad BIOC 463A Expt 4 Column Chromatographic Methods The Voumes of SEC The degree of interaction of the protein with the SEC resin therefore its ability to separate proteins of different sizes or masses is best measured by the VOLUME in which the sample elutes from the column Volume Definitions 0 V0 Void Volume the volume outside of the beads 0 Vi Included Volume the volume within the beads 0 V9 Gel Volume the volume of the beads themselves S 1 of total volume 0 Vtotal Total Volume of column VtVoViVg Since Vg 0 then Vt V0 Vi 0 V9 Elution Volume for a sample How are these volumes measured Referring to Fig 44 the volumes are calculated from the fraction of a known volume at which the material elutes from the column 10 BIOC 463A Expt 4 Column Chromatographic Methods Fig 44 A B 0 059 O O O O 0 Volume Void Volume V0 is determined using a material usually a colored dye such as BLUE DEXTRAN that is too large to interact with the resin therefore is not retarded by the resin as it flows through the column O 0 0390 00 O 000 GO 000 O Total Volume Vt is determined by using a material that is very small and interacts maximally with the resin As in the above case often a colored inorganic salt FERRICYANIDE OR POTASSIUM CHROMATE can be used We will use FerriCyanide FeCN5339 11 BIOC 463A Expt 4 Column Chromatographic Methods The volume at which this material elutes includes both the void outside and included inside volumes Partition Coefficient Kav Every protein or DNA or RNA elutes from a specific SEC column in a reproducible manner based on its Stokes Radius which is related to its mass The measure of this elution behavior is expressed as its Partition Coefficient Kav Kav Ve V0Vt Vo Ve VoN i 12 BIOC 463A Expt 4 Column Chromatographic Methods Calibration of SEC Columns The knowledge of the Kav for a given protein can be very useful in the determination of the molecular weight or mass of a given protein in the following manner 1 A precise volume of a mixture of proteins Blue Dextran and a small molecular weight indicator are eluted from the column and their Kav values determined 2 A plot of Kav vs Log Mol Weight MW is constructed 3 The unknown protein is added to the column using the same volume as used for standards then eluted from the column The Kav is determined for the unknown and MW is calculated from the plot of Kav vs Log MW 13 BIDC 4A Expt 4 Column Chromatographic Methods Fig 45 Kav vs Log MW Ln 77 quot c e B o a 7 4 5 w 4 a 4 02 u L L moo 104 105 10 MW Kav f 39 14 BIOC 463A Expt 4 Column Chromatographic Methods Resolution of SEC Columns 0 Resolution is proportional to Length1 2 But elution time is also proportional to length gt YOU MUST BE PATIENT WHEN RUNNING COLUMNS o The shallower the slope of a Kav vs Log MW plot ie the broader the range of MWT resolved the greater the resolution of the column resin Plot of Kav vs Log MW for G75 G100 and 6200 15 BIOC 463A Expt 4 Column Chromatographic Methods Problems with SEC 0 Initial equilibration is long and tedious Involves hydration swelling then pulling a vacuum on the resin to remove air from within beads o CANNOT allow resin to go dry If so then repeat equilibration process 0 For sugar based resins algae and bacteria can grow on sugar matrix Store using a 02 NaAzide solution 0 Packing of column is critical Best if done in continuous manner so all of resin settles at same time Eliminates banding in column Sephadex resins Volume decreases with increase in ionic strength 0 Flow rate decreases with increase in MW range Le 6200 runs slower than 625 Can compensate by using peristaltic pump BUT 0 High pressure can collapse Sephadex beads at bottom of column use polyacrylamide resins Bands are broadened on SEC columns sample is diluted on SEC columns due to thermal diffusion and frictional effects Improperly poured columns or columns with 16 BIOC 463A Expt 4 Column Chromatographic Methods plugged frits can result in very erratic band migration 0 Air bubbles can form in column when taken from cold room to room temp due to expansion of gas volume Room temp 9 cold OK Cold room 9 room temp Bad 17 BIDC 463A Expt 4 Column Chromatographic Methods Fig 46 Proper Loading of SEC Columns Use caution so the surface of the column is not disturbed 18 BIOC 463A Expt 4 Column Chromatographic Methods Ion Exchanqe Chromatoqraphv DEAE and CMC Uses 1 Crude purifications of cell lysates ie batch cuts 2 Decrease volume of protein sample ie concentrate 3 Purify proteins according to charge properties of protein and buffer conditions pH and ionic strength Separation based on net charge on protein and electrostatic interactions between protein and charged groups on resin Positively Charged AA Negatively Charged AA Arg Asp Lys Glu His RS39 Znet Zpos Zneg Znet is related to pH and pl pH lt Znet gt 0 pH pl Znet 0 pH gt Znet lt 0 19 BIOC 463A Expt 4 Column Chromatographic Methods Often proteins are referred to as Acidic Basic or Neutral Acidic lots of Asp pllt6 Glu Neutral 6 lt pl lt 8 Basic lots of Arg plgt8 Lys Strong vs Weak Ion Exchangers see Table 42 for names and structures Strong Used to remove small anions or cations Irreversible binding of ions to resin Often used for stripping purposes Weak Reversiny binds anions or cations ie proteins with negative or positive net charge 20 B IOC 463A Expt 4 Column Chromatographic Methods TABLE 42 Ion Exchange Groups Used in the Purification of Proteins FORMULA NAME ABBREVIATION Strng anion iCHzNWCHSB lrimelhylmninoethyl TAM iCzHJNWCZHSh H iclh luminoethyl TEAE 7C3H4NiC3P152CH1 HOHJCH3 dielhylZhydroxypropyumino clhyl QAE Weak anion 7C3H4NH3 uminoelhyl AE 1114NHC2H53 dielhyluminoelhyl DEAE Sirmg cation 7803 sulpho S iCHESQ sulphomethyl SM 7C3HbSO3 sul phopropyl SP Weak cation 7COO carboxy C iCHICOO carboxymelhyl CM 21 Expt 4 Column Chromatographic Methods Fig 411 How IEC Columns Bind Proteins 590 a rmua a Hhm mhun a mexcmnge pmcessas accumwg Wu neaa ne y havgcd quotmenu ar spamVerJ on m mmmcmngn calumn m comm 5 mmnmm v man a mom as mam By W VNACIJ mm mm m Ina mallFX mm 3 Dr and Na mm mmch asscr mam wmm mm a m mm a As was movesses ma Lss ammo munwmw mnghqleaClJ lhe 39namacnhnpmrm Le ulcs 22 BIOC 463A Expt 4 Column Chromatographic Methods IEC Protocol Resin Equilibration Very important in order to maximize binding of proteins These steps are usually done in a beaker that allows you to use buffer or solution volumes 5X the resin volume 0 Initial equilibration Cycle resin with 01 M NaOH and 01 M HCI or other acid for 5 min exposure timeaddition o Equilibrate with high buffer at correct pH 0 Equilibrate with decreasing buffer ending at desired concentration at correct pH With each addition the resin is gently stirred then allowed to settle The excess solution is then poured off This also helps remove broken resin particles or FINES which tend to plug up frits at the bottom of the column This process is repeated until the ionic strength ie concentration measured using a conductivity meter and the pH measured using a pH meter are at the desired values In almost every case ion exchange resins are equilibrated at very low ionic strength thus allowing maximal electrostatic interaction between your proteins and the resin 23 BIOC 463A Expt 4 Column Chromatographic Methods Protein Loadinq and Elution Typically IEC columns are short amp large diameter in order to maximize flow rates Equilibrated resin is poured into column as a slurry and rinsed down the sides of the glass column using the equilibration buffer Note DEAE resins containing a positively charged group interact with glass silica oxides annoyingly well and require diligent rinsing to get all of the resin down to the bottom of the column Negatively charged CMC resins do not have this problem Once the column is poured a set amount of equilibration buffer is passed through the column with the effluent being checked for both conductance and pH Unlike SEC chromatography proteins can be loaded onto IEC columns at dilute concentrations In fact one of the purposes of IEC resins is to CONCENTRATE your protein It is easiest to have all of your protein in a beaker that is connected to the column by a siphoning gravity feed tube 24 BIOC 463A Expt 4 Column Chromatographic Methods Once all of the protein solution has been loaded onto the column a specified volume of LOW IONIC STRENGTH buffer is passed through the column in order to rinse off any proteins that are not binding to the column via electrostatic interactions Elution of Proteins There are two commonly used ways to elute the proteins from an IEC column depending on the intentions of the researcher Batch elution or cut If the goal of the column is to primarily CONCENTRATE a large volume of protein down to a smaller volume then ALL of the proteins are eluted off using a single step method called a BATCH ELUTION or CUT In this method a sufficiently HIGH IONIC STRENGTH buffer is added to strip ALL PROTEINS bound to the column Purification by this method is minimal however you do get rid of proteins that have the same sign of charge as the resin Batch cuts are often done very early in a purification scheme for instance immediately after a cell lysis step The main 25 BIDC 4A Expt 4 Column Chromatographic Methods goal is to CONCENTRATE or DECREASE the VOLUME of your sample High resolution IEC Chromatography If the goal of the column is to achieve purification then the proteins are eluted off the column in a gradual manner typically using an IONIC STRENGTH GRADIENT omnaw Tube Erldge HIGH SALT An Ionic Strength Gradient is established as the column siphons buffer from the Out ow Beaker which in turns siphons from the High Salt Beaker Mixing the High Salt with the Low Salt solution gradually increases the ionic strength creating a linear gradient owing onto the column 26 BIOC 463A Expt 4 Column Chromatographic Methods Another Way to Elute Proteins from IEC Columns 0 IEC columns bind proteins via electrostatic interactions between the resin and protein 0 The SIGN and AMOUNT of charge on protein is related to pl a fixed property of protein and pH variable 0 Charge on protein can be diminished by adjusting pH towards pl when pH pl the Zprotein 0 Instead of using IONIC STRENGTH gradient can use a pH gradient instead 27 BIDC 4A Expt 4 Column Chromatographic Metnods 2 L cell lysate DEAE Cellulose Load 8t Rinse Low buffer Elute WI High buffer Elution Vol 100 mL Sephadex 3100 Load 100 rnL Fraction Vol 10 rnL a Fractions pooled to further purify DEAE Cellulose 0R Load amp Rinse Lowl Ammty C quotquotquotquot39 Elute so 500 mm Pi gradient Load and rinse Elute wI Substrate or Ligand analog This schematic shows a typical purification procedure using a combination of SEC IEC and affinity columns Which steps are used to concentration OR separate OR concentrate AND separate 28 BIOC 463A Expt 4 Column Chromatographic Methods Column Chromatography Part 2 Affinity Chromatography There are three parts to an affinity resin 0 An activated resin has reactive sites 0 Activated Linker of defined length 0 Ligand or Substrate analog inert that can be bound by linker General Procedure Figure 47 Affinity chromatography A B L R QW OWE QM R ow R l R 29 BIDC 4A Expt 4 Column Chromatographic Methods Affinity column resins can be purchased pre made or you have to carry out the following reaction sequence Ligand or Sanalog A Activated Af nity Resin 30 BIOC 463A Expt 4 Column Chromatographic Methods Advantages of Affinity Chromatography Interactions are highly specific for ReceptorLigand or EnzymeSubstrate complexes hydrophobic van der Waals hyd Bonding o Simplifies purification schemes dramatically Onestep purification claims by manufacturers are overstated Disadvantages o If affinity resin not available must follow procedure to prepare resin Resins seem to have finite lifetime especially Substrate analog resins They lose effectiveness with time Special Consideration You do not want to use a Ligand or Substrate that has such a low K or Ks that it is difficult to remove from column 31 BIDC 4A Expt 4 Column Chromatographic Methods Affinity Column Purification of 3galactosidase W l H qua W W Kw x l 2 l V OH I l u i wlzL39H Luciusu l n Baum m u n Glumm FIGURE 414 Reaction catalyzed by ligalaclosmass We will purify 3galactosidase making use of the fact that it has a high degree of specificity for binding galactose w HO on H0 Againsev L 1 MW p e v W ut NVW 1 I f mm a paminobenzyl1thioBDgalactopyranoside agarose resin Why a Sglyscosidic linkage instead of an Oglycosidic linkage 32 BIDC 4A Expt 4 Column Chromatographic Methods TAG or Gene Fusion Columns A Short Primer on the Recombinant Protocol Restriction Circular Enzyme P39asmi gt Plasmid Multiple Cloning I 39 Gene Site Ligation Lots of Protein Q g 1 g 1 g E coli Gene Expression 33 BIOC 463A Expt 4 Column Chromatographic Methods General Principle TAG a short peptide or protein that will interact very strongly with an affinity like column 0 The DNA sequence for the TAG is expressed at either the N or Cterminus of your protein of interest 0 Interaction of the TAG with the column that is specific for the TAG supposedly allows for a high yield 1step purification o Tagging plasmid and Column resin are sold by same manufacturer 34 BIDC AA Expt 4 Column Chromatographic Methods Maltose Binding Protein Tag hepmu m m mm I m M m m squot m m Manamammn mmmmmmmmmmmmmnanm I at m In mumm 35 BIOC 463A Expt 4 Column Chromatographic Methods Common Tagging Systems Gene Fusion Column Maltose Binding Maltose analog Protein Glutathione S Glutathione Transferase Peptide Taq Column PolyHis 78 Chelated Nickel Avidin Binding Streptavidin Pep de Despite the attractiveness of Gene Fusion systems there are concerns one should be aware of with these constructs 36 BIOC 463A Expt 4 Column Chromatographic Methods The goal of the protein biochemist is to understand how a NATIVE protein functions in the NATIVE organism But with Tagged systems we have Native vs Wildtype original source recombinant Wildtype vs Tagged not cleaved Wildtype vs Cleaved tagged Cleaved vs Native Although it is often done it is DANGEROUS to ASSUME we all know what ASSUME means don t we that any of the recombinant proteins wild type tagged not cleaved or cleaved will behave EXACTLY like the native protein ALWAYS DO A DIRECT COMPARISON 37
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