Quantitative immunoblot detection of rare proteins in whole cell extracts using biotin-strepavidin reagents.
A novel immunoblotting method designed for quantitative detection of low copy-number proteins in crude cell extracts is presented. This technique can be used with either mono- or poly-clonal antibodies and utilizes the sensitivity and amplification of the high affinity interaction between biotin and strepavidin. Radioactive iodination of the strepavidin moiety allows for rapid and accurate quantification of proteins bound to nitrocellulose. This biotin/125I-strepavidin technique is highly reproducible and can detect as little as 1 ng of protein. In addition, use of biotinylated secondary antibodies directed against a specific type of primary antibody avoids the problem of low affinity recognition of immunoglobulins from certain species by protein A. Finally, the methodology is simple and convenient, and secondary and tertiary reagents are commercially available. The application of this technique is illustrated in the determination of relative quantities of the tight junction-associated protein ZO-1, present in very small amounts in epithelial cells. This same technique can also be used for the quantitative analysis of relatively more abundant cellular constituents or purified protein.
Stevenson
B R
BR
Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Canada.
Richards
C L
CL
Howarth
A G
AG
Maraj
V A
VA
Hibbard
J G
JG
D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000032:analysis*; D051180:Desmoplakins; D004285:Dogs; D015151:Immunoblotting / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D019809:Streptavidin; D062826:Zonula Occludens-1 Protein
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D001710:Biotin; D019809:Streptavidin