Affinity Blotting

Covering the blotting membrane with a suitable capture ligate, e.g., antibody, increases the sensitivity for detecting the corresponding ligand, e.g., antigen. 

Dilute the affinity-purified capture antibody to lO-lOOpg/ml in Soln. A. Incubate a new piece of blotting membrane with the antibody dilution (0.2-0.5 ml/cm ) at RT for 30 min. 

To save antibody, use a sealable tube and a roller shaker. If such a tube is used, make sure that the receiving side of the membrane is directed to the lumen of the tube. 

Wipe off remaining antibody dilution after incubation with filter paper and incubate the wet membrane in Soln. B for 30 min. Rinse with Soln. C and stop the fixation by incubation with Soln. D for 2 min. Block with Soln. E for 30 min, wash three times with Soln. C, and dry in vacuo. 

The activated dry membrane can be stored m a refrigerator for several weeks. 

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Estrada, E., et al. (1991). Identification of the Vasopressin Receptor by Chemical Crosslinking and Ligand Affinity Blotting, Biochemistry 30 8611-8616. 

In a preliminary report, Ross et al. [40] used affinity chromatography to identify a putative bovine renal brush border Na /H exchanger. Brush border membranes were solubilized with Triton X-100 and chromatographed sequentially over lentil lectin Sepharose 4B and 5-(A-benzyl-iV-ethyl)amiloride coupled to epoxy-activated Sepharose 6B. The eluant contained 178- and 146-kDa proteins that were susceptible to Endo-F. Moreover, the eluants reacted on dot blot immunoassays with antisera to a 20-amino acid peptide of a human Na /H exchanger vide infra). The relationship between these proteins and the 66-kDa protein previously identified by the same investigators using amiloride photolabeling is presently unclear. 

The affinity and specificity of the antibodies used will have a great impact on the sensitivity of a blotting system, and the choice between monoclonal or polyclonal antibodies must be made (refer to Table 8-1). An overview of the variety of approaches that can be taken to isolate proteins for antibody production can be found in Diano et al. (1987), Leppard et al. (1983), and Knudsen (1985). 

For many years, due to the availability and low cost of radioisotope-labeled secondary antibodies, radioactive detection was the method of choice in Western blotting. Newer methods that are less hazardous and easier to use, while maintaining comparable sensitivity, have been developed. Today, Western blotting detection methods can be light-based, (chemiluminescence, bioluminescence, chemifluorescence, and fluorescence), radioactivity-based, or color-based. It is important to note that the detection sensitivity depends on the affinity of the primary antibody for the antigen and on the affinity of the secondary antibody for the primary antibody and can therefore vary considerably from one protein sample to another and from one antibody batch to another. 

Blot the proteins onto the affinity membrane either by dot blot (Protocol 4.8), by electrotransfer (Protocol 2.5.3), or by capillary blotting (handling analogously to Southern blotting Protocol 2.5.9). 

CapiUary blotting is recommended, if the gel is mounted onto a impermeable carrier. Put the affinity membrane with the coated face towards the gel onto the gel, cover the backside of the membrane with several layers of dry filter paper, wrap the stack into a water-tight foil, and store it under moderate pressure in a refrigerator for several hours or overnight. 

The principal limitation of these data is the lack of definition of the individual forms for the CYP2C subfamily. Analysis of this subfamily has remained problematic due to high cross-reactivities of all of the distinct forms with most antibody preparations. In addition, Western blot analysis does not distinguish between active and inactive forms of the protein. Furthermore, distinct enzymes may have different affinities for coenzymes necessary for catalytic activity, which will serve to unlink abundance of the protein and its catalytic activity. Therefore the assumptions must be made that the ratios of active to inactive protein are similar for all forms and that all forms have similar affinities for coenzymes. These assumptions may not be justified. However, even with these limitations, the study of Shimada et al. (1994) contributes greatly to our understanding of relative enzyme abundance in human liver. In addition, the relative abundance data, coupled with the absolute P450 content (per unit protein) and the turnover numbers for enzyme-specific substrates (per unit protein), can provide an estimate of the turnover number for individual enzymes in the human liver membrane environment. This provides an important benchmark for evaluation of turnover number data from cDNA-expressed enzymes. 

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