Protein purification affinity chromatography

Besides protein microarray proteome profiling, the term chemical proteomics (also chemoproteomics or pull-downs ) is mostly used in reference to the application of affinity chromatography protein purification when small molecules are the bait, and liquid chromatography separation of peptides precludes mass spectrometry as the universal readout (LC-MS/MS). 

Porath, J. (1992) Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263-281. 

A2. Allen, R. H., and Mehlman, C. S., Isolation of gastric vitamin B12 binding proteins using affinity chromatography. I. Purification and properties of human intrinsic factor. /. Biol. Chem. 248, 3660-3669 (1973). 

Antibody Purification Affinity Chromatography - Protein A and Protein G Sepharose... 

Allen, R.H., Majerus, P.W. Isolation of vitamin Bi2-binding proteins using affinity chromatography II. Purification and properties of a human granulocyte vitamin B 12-binding protein. J. biol. Chem. 247, 7702-7708 (1972)... 

The protein under study should be used in homogeneous solution if possible. Fortunately, affinity chromatography makes purification and electrophoretic diffusion makes even crystallization of such enzymes possible. 

Vorackova 1, Suchanova S, Ulbrich P, Diehl WF, Ruml T. Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography. Protein Expres. Purif 2011 79 88-95. 

PCCase is a biotinylated protein that catalyzes a reaction required in the catabolism of amino acids and fatty acids of odd-numbered chain length, and in the catabolism and anabolism of branched-chain fatty acids. In order to characterize the structure of this enzyme from plants we undertook its purification. PCCase activity was purified from extracts of maize leaves by a four step scheme that included PEG precipitation, hydrophobic interaction chromatography, anion exchange chromatography and affinity chromatography. This purification scheme achieved a nearly 250-fold purification of PCCase activity. However, throughout this purification of PCCase, ACCase copurified. Indeed, SDS-PAGE analysis of the final purified PCCase preparation identified two biotinylated polypeptides of about 240 and 230 kDa. These polypeptides have previously been described as subunits of ACCase (7). Furthermore, mixed substrate kinetic studies (8) with the purified PCCase/ACCase preparation indicated that the carboxylation of propionyl-CoA and acetyl-CoA were carried out by the same enzyme. Furthermore, both PCCase and ACCase activities were similarly affected by a variety of inhibitors. 

Acyl-ACP thioesterase activity was assayed as described [1]. All purification steps were carried out at 4°C. Two hundred grams of frozen leek epidermis were ground to a powder in liquid nitrogen using a prechilled mortar and pestle. The protein was precipitated with 75% (w/v) ammonium sulfate, and then subjected to hydroxyapatite, Mono-S, Mono-Q, and ACP affinity chromatography. Proteins from each purification step were separated by SDS polyacrylamide gel electrophoresis according to Laemmli (1978)[3] and silver stained (Bio-Rad). 

In many cases, proteins can be modified with affinity tags (e.g., polyhistidine), which arc fused to the protein via molecular genetic manipulations to facilitate purification by using affinity chromatography. Proteins modified with histidine residues are isolated from mixtures by using the polyhistidine motifs affinity for nickel or cobalt as the selective step. The modification is made by making genetic... 

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