QUANTITATIVE IMMUNOBLOT DETECTION OF RARE PROTEINS IN WHOLE-CELL EXTRACTS USING BIOTIN-STREPAVIDIN REAGENTS

A novel immunoblotting method designed for quantitative detection of l ow copy-number proteins in crude cell extracts is presented. This tech nique can be used with either mono- or poly-clonal antibodies and util izes the sensitivity and amplification of the high affinity interactio n between biotin and strepavidin. Radioactive iodination of the strepa vidin moiety allows for rapid and accurate quantification of proteins bound to nitrocellulose. This biotin/I-125-strepavidin technique is hi ghly reproducible and can detect as little as 1 ng of protein. In addi tion, use of biotinylated secondary antibodies directed against a spec ific type of primary antibody avoids the problem of low affinity recog nition of immunoglobulins from certain species by protein A. Finally, the methodology is simple and convenient, and secondary and tertiary r eagents are commercially available. The application of this technique is illustrated in the determination of relative quantities of the tigh t junction-associated protein ZO-1, present in very small amounts in e pithelial cells. This same technique can also be used for the quantita tive analysis of relatively more abundant cellular constituents or pur ified protein. (C) 1994 Wiley-Liss, Inc.