ZFIN ID: ZDB-PUB-140513-300
Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system
Ahmed, R., Spikings, E., Zhou, S., Thompsett, A., Zhang, T.
Date: 2014
Source: Journal of immunological methods 406: 143-7 (Journal)
Registered Authors: Zhang, Tiantian
Keywords: Biotinylated secondary antibody, Endogenous biotin binding, Non-specific bands, Qdot 625 streptavidin conjugate, Streptavidin, Western blot
MeSH Terms:
  • Actins/immunology
  • Animals
  • Antibodies/immunology
  • Biotin/immunology*
  • Biotinylation/methods
  • Blotting, Western/methods*
  • Humans
  • Streptavidin/immunology*
  • Zebrafish/embryology*
PubMed: 24657589 Full text @ J. Immunol. Methods
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ABSTRACT
Endogenous biotin or biotinylated protein binding activity is a major drawback to biotin-avidin/streptavidin detection system. The avidin/streptavidin conjugate used to detect the complex of the biotinylated secondary antibody and the primary antibody binds to endogenous biotin or biotinylated proteins leading to non-specific signals. In Western blot, the endogenous biotin or biotinylated protein binding activity is usually manifested in the form of ~72kDa, ~75kDa and ~150kDa protein bands, which often mask the signals of interest. To overcome this problem, a method based on prior hybridisation of the biotinylated secondary antibody and the streptavidin conjugate was developed. The method was tested alongside the conventional biotin-streptavidin method on proteins extracted from zebrafish (Danio rerio) embryos. Results showed that the newly developed method efficiently suppresses the endogenous biotin or biotinylated protein binding activity inherent to the biotin-streptavidin detection system.
ADDITIONAL INFORMATIONNo data available