Troubleshooting affinity chromatography
Contents
1. 55 Zhou Q Lieberman P M Boyer T G and Berk AJJ 1992 Holo TFIID supports transcriptional stimulation by diverse activators and from a TATA less promotor Genes and Development 6 10 1964 1974 lt q CONTENTS2. 3 Dean P D G J ohnson W S and Middle F A 1985 Affinity Chromatography A Practical Approach IRL Press Field J Nikawa J I Broek D MacDonald B Rodgers L Wilson A Lerner R A and Wigler M 1988 Purification of a RAS responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method Mol Cell Biol 8 5 2159 2165 Frost R G Monthony J F Engelhorn S C and Siebert C J 1981 Covalent immobilization of pro teins to N Hydroxysuccinimide ester derivatives of agarose Effect of protein charge on immobilization Biochim Biophys Acta 670 2 163 169 Harlow E and Lane D 1988 Antibodies A Laboratory Manual Cold Spring Harbor N Y Press Hermanson G T Mallia A K and Smith P K 1992 Immobilized Affinity Ligand Technologies Academic Press O Shannessy D J and Hoffman W L 1987 Site directed immobilization of glycoproteins on hydrazide containing solid supports Biotech Appl Biochem 9 5 488 496 Prickett K S Amberg D C and Hopp T P 1989 A calcium dependent antibody for identification and purification of recombinant proteins BioTechniques 7 6 580 589 Templeton D J 1992 Nuclear binding of purified retinoblastoma gene product is determined by cell cycle regulated phosphorylation Mol Cell Biol 12 2 435 443 Wilchek M Miron K T and Kohn J 1984 Affinity chromatography Methods Enzymol 104 3
3. Immunoaffinity Purification Affinity chromatography Troubleshooting affinity chromatography Problem Tagged protein appears in washes does not bind to affinity resin Possible cause Remedy Antibody did not couple to support resin Try another method to couple the antibody to the resin Antibody binding site altered or blocked Use a different chemical technique to couple during antibody resin coupling procedure the antibody to the resin For example couple cannot bind tagged protein the antibody through its carbohydrate groups hydrazide coupling rather than through its amino groups One or more of these sample loading Do trial experiments to optimize loading conditions may not be optimal for parameters Follow these general guidelines binding tagged protein to affinity column pH e Load at neutral pH e Flow rate e Load sample slowly or use batch loading e Temperature e Load at room temperature e Salt or ion concentration e Salt and ion concentration in Binding Buffer should be kept low Proteases are degrading tagged protein Add protease inhibitors BM to sample loading wash buffer Note See Section 4B of this manual for more infor mation on protease inhibitors Antibody on affinity column was Always wash column with Binding Buffer right inactivated during prior use after each use Always store column at 4 C in Binding Buffer Azide Prepare a new column Channels have formed in column bed Re p
4. ack column so loaded sample runs through column without interacting with antibody Problem Tagged protein remains bound to column does not elute in Elution Buffer Possible cause Remedy Elution Buffer is not optimal Try alternative elution buffers such as described in Procedure Il Try a combination of the elution buffers described in Procedure II Try elution at a higher temperature lt q CONTENTS Immunoaffinity Purification Affinity chromatography Problem Extra proteins in eluant with tagged protein Possible cause Remedy Nonspecific proteins binding to affinity resin Before applying sample the first time block the resin with a protein that will not later interfere with the chromatography for instance bovine serum albumin After sample application wash column more thoroughly before eluting tagged protein Wash columns with a more stringent second Wash Buffer for example with higher salt Apply sample in a Binding Buffer containing enough salt to minimize nonspecific binding but not the binding of the tagged protein Problem Tagged protein degraded Possible cause Remedy Proteases in sample Include protease inhibitors throughout procedures Work at low temperature such as 4 C to minimize degradation Tagged protein unstable Work at low temperature such as 4 C to minimize degradation Problem Bubbles in affinity column Possible cause Remed
5. y Column poured and stored at one temperature but used at another Always allow column to equilibrate to procedure temperature then degas under vacuum and remix resin to remove bubbles before using OR Pour column at the same temperature that it will be used OR Pour column at a warmer temperature then let it equilibrate to a cooler temperature Use the column at the cooler temperature Problem Tagged protein elutes as a diffuse band Possible cause Remedy Elution Buffer does not immediately release protein from resin Increase concentration of release agent peptide salt etc in Elution Buffer Elution causes band diffusion Use reverse upward flow to elute the column Note requires mechanical pump to create upward flow lt q CONTENTS 4 13 4 14 Suggested reading to learn more about procedure There are numerous affinity purification protocols described in the literature For a detailed description of these techniques and discussion of factors affecting the results see Hermanson Mallia and Smith 1992 Other helpful references include Cuatrecases Wilchek and Anfinsen 1968 Dean Johnson and Middle 1985 Harlow and Lane 1988 and Wilchek Miron and Kohn 1984 Immunoaffinity Purification Affinity chromatography References Cuatrecasas P Wilchek M and Anfinsen C B 1968 Selective enzyme purification by affinity chromatog raphy Biochemistry 61 636 4
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