Affinity chromatography elution procedures

Fidder and coworkers (50) developed a versatile procedure that identifies phosphylated butyrylcholi-nesterase. Adducted butyrylcholinesterase is isolated from plasma by affinity chromatography (procainamide column), digested with pepsin, and a nonapep-tide containing the phosphylated active-site serine residue detected using LC/ESI/MS/MS (quadrupole-TOF hybrid instrument). A C18 150 x 0.3-mm LC column was used, eluted with a gradient of water-acetonitrile-0.2 % formic acid. The method was applied successfully to casualties of sarin poisoning from the Tokyo subway attack (see Chapter 17). 

In practice, affinity chromatography is usually designed to be the final (if not the sole) chromatographic step in a purification procedure, so as to protect the valuable affinity material from unnecessary contamination. It is normal to use short columns whose capacity is largely fully exploited. Elution can be by a batch process or gradi-... 

Affinity chromatography is carried out by the batch and column methods. The procedure involves (i) equilibration of the adsorbent, (ii) preparation of sample, (iii) application of the sample, (iv) washing away of unbound materials, (v) elution and (vi) regeneration of adsorbent. 

The use of amylose gel cross-linked by epichlorohydrin for affinity chromatography of extracellular isoamylase of Pseudomonas amyloderamosa has been studied. The isoamylase was adsorbed well on the amylose gel and was eluted specifically with maltodextrin. The eluted enzyme was precipitated with ammonium sulphate to remove maltodextrin, and then the solution of the precipitate was dialysed and concentrated by vacuum filtration. By this procedure 96 mg of the enzyme were purified to homogeneity from 20 1 of culture broth in about 70% yield. 

Rad Laboratories) according to procedures recommended by the manufacturer. After the concentration of pooled fractions from the last purification step (Fractogel column) by ultrafiltration, affinity chromatography was used. Partially purified PQC was injected into a 12.5 X 1.6 cm inner diamater column of immobilized trypsin gel preequilibrated in the eluting buffer (20 mM in 50 mM Tris-HCl buffer, pH 8.2). Affinity purification was accomplished because PQC contains a unique and highly basic polypeptide chain containing covalently attached phosphate groups without any disulfide bonds. 

An essentially pure extracellular glycoprotein proteinase inhibitor was isolated from the latex of green fruits of papaya by a single affinity chromatography purification step. An immobilized trypsin-Sepharose CL 4B column was prepared according to the manufacturer (Amersham Pharmacia Biotech), which provided elaborated bulletins for the preparation procedures and applications of these affinity gel columns. Latex extract was applied to the column after equilibration with 20 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl and 0.05 M CaCli. The column was extensively washed with the same buffer until ultraviolet (UV) absorbance became undetectible. The bound trypsin inhibitor was eluted with 0.02 M HCl and recovered by lyophilization after dialysis against water. 

Qualitative analysis of the eluted fractions was carried out by silver staining. We have utilized a procedure that is optimized to allow analysis by MALDITOF microsequencing of the stained bands, if required (Shevchenko et al, 1996). If the preparation has to be used for microsequencing, the isolation by affinity chromatography on Rac-GTP matrix should be scaled up by a factor of 5 to 10 (referred to as larger preparations in the previous paragraph). 

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