Avidin blotting

Figure 1. Immunoblots and avidin blots of recombinant E, coli DH5ocF cells expressing C. cryptica ACCase. Lane A - Control cells containing pBluescript KS+. Lane B - pACC20M9-containing ceUs. Lane C - purified C. cryptica ACCase. Secondary antibodies and avidin were conjugated to alkaline phosphatase. The gels used for these blots were loaded with equal amounts of protein.

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

Bayer, E.A., Ben-Hur, H., and Wilchek, M. (1987a) Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology. Anal. Biochem. 161, 123-131. 

Results from a Western blot. A SDS-PAGE gels, 12%, were run and transferred to nitrocellulose. Lane 1, MW standards lane 2, biotinylated standards lane 3, human transferrin lane 4, . coli lysate lane 5, total human serum lane 6, biotinylated standards. Gel A was stained with a protein dye. Blot B was assayed using rabbit anti-human transferrin as the first antibody. The second antibody solution contained anti-rabbit HRP conjugates. Only the transferrin bands and the prestained biotinylated standards were detected by the antibodies and the avidin-HRP treatment. 

Figure B3.4.1 Serial dilution of primary antibody directed against the 97-kDa catalytic subunit of the plant plasma membrane ATPase. The blot was developed with HRP-coupled avidin-biotin reagents according to the Alternate Protocol and visualized with 4-chloro-1-naphthol (4CN). Note how background improves with dilution...

Hybridization in a plastic bag requires 5 to 10 ml avidin-biotin-enzyme solution. Membrane strips require 5 to 10 ml/strip, whereas blots from standard-sized gels (i.e., 14 x 16 cm) require 50 ml for convenient handling in a tray. 

Avidin-gold or streptavidin-gold conjugates can be used to detect, localize, or quantify the binding of biotinylated molecules in cells, tissue sections, or blots (Morris and Saelinger, 1984 Bonnard et al., 1984 Gillitzer etal., 1990 Bronckers et al., 1987)... 

To examine the purity of the protein that was eluted from a Sephacryl S-300 column, we subjected the protein to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and then performed a Western blot (26) using avidin linked to phosphatase as a probe. The SDS-PAGE indicated that there were many polypeptides in this partly purified preparation (Fig. 6). Among these were two major and several minor biotin-containing bands (Fig. 7). This preparation may contain propionyl CoA carboxylase or a partly degraded form of ACCase that can use propionyl CoA as well as acetyl CoA as a substrate. Therefore, the enzyme data presented here are quali-... 

The K138C mutants (-1.5 mg in 0.5 mL of PBS) were treated with 50 mM DTT (removed by gel filtration) and then biotinylated using biotin-maleimide (Sigma) (2 1 molar ratio). The biotinylated product was purified by gel filtration on Superdex 75. Evidence of biotinylation was routinely obtained by western blots probed with an avidin-HRP conjugate (Pierce) and by binding of the proteins to streptavidin-coated BIAcore chips (Pharmacia SA5). 

In addition to the detection of antigens and antibodies, EIA will, undoubtedly, play an increasingly important role in molecular biology. For example, the bio-blot method (Leary et al., 1983) for the detection of DNA-DNA or DNA-RNA duplexes on nitrocellulose membranes offers important advantages over conventional procedures in which radioactive probes are used and autoradiographic detection. In this method, biotinylated DNA probes are prepared by nick translation (Rigby et al., 1977) in the presence of biotinylated analogs and hybridized with the DNA or RNA on filters. Biotin is then detected by avidin-labeled enzyme (Section 3.1). 

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