title: Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system.
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.
authors.affiliation: Institute of Biomedical and Environmental Sciences and Technology (iBEST), University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire LU2 8DL, UK. Electronic address: raju79@hotmail.co.uk.
authors.forename: Raju
authors.initials: R
authors.lastname: Ahmed
authors.affiliation: Institute of Biomedical and Environmental Sciences and Technology (iBEST), University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire LU2 8DL, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Spikings
authors.affiliation: Institute of Biomedical and Environmental Sciences and Technology (iBEST), University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire LU2 8DL, UK.
authors.forename: Shaobo
authors.initials: S
authors.lastname: Zhou
authors.affiliation: School of Health, Sport and Bioscience, University of East London, Stratford Campus, London E15 4LZ, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Thompsett
authors.affiliation: School of Applied Sciences, Bournemouth University, Poole, Dorset BH12 5BB, UK.
authors.forename: Tiantian
authors.initials: T
authors.lastname: Zhang
mesh_terms: D000199:Actins / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001710:Biotin / Q000276:immunology*; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western / Q000379:methods*; D006801:Humans; D019809:Streptavidin / Q000276:immunology*; D015027:Zebrafish / Q000196:embryology*
chemical_list: D000199:Actins; D000906:Antibodies; D001710:Biotin; D019809:Streptavidin
keywords: Biotinylated secondary antibody; Endogenous biotin binding; Non-specific bands; Qdot 625 streptavidin conjugate; Streptavidin; Western blot