Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system

Authors: Ahmed, R., Spikings, E., Zhou, S., Thompsett, A. and Zhang, T.

Journal: Journal of Immunological Methods

eISSN: 1872-7905

ISSN: 0022-1759

DOI: 10.1016/j.jim.2014.03.010

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 ~ 72 kDa, ~ 75 kDa and ~ 150 kDa 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. © 2014 Elsevier B.V. All rights reserved.

Source: Scopus

Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system

Authors: Ahmed, R., Spikings, E., Zhou, S., Thompsett, A. and Zhang, T.

Journal: Journal of Immunological Methods

Volume: 406

Pages: 143-147

eISSN: 1872-7905

ISSN: 0022-1759

DOI: 10.1016/j.jim.2014.03.010

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 ~. 72. kDa, ~. 75. kDa and ~. 150. kDa 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. © 2014 Elsevier B.V.

Source: Scopus

Preferred by: Tiantian Zhang

Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system.

Authors: Ahmed, R., Spikings, E., Zhou, S., Thompsett, A. and Zhang, T.

Journal: J Immunol Methods

Volume: 406

Pages: 143-147

eISSN: 1872-7905

DOI: 10.1016/j.jim.2014.03.010

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.

Source: PubMed

Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system

Authors: Ahmed, R., Spikings, E., Zhou, S., Thompsett, A. and Zhang, T.

Journal: JOURNAL OF IMMUNOLOGICAL METHODS

Volume: 406

Pages: 143-147

eISSN: 1872-7905

ISSN: 0022-1759

DOI: 10.1016/j.jim.2014.03.010

Source: Web of Science (Lite)

Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin-streptavidin detection system.

Authors: Ahmed, R., Spikings, E., Zhou, S., Thompsett, A. and Zhang, T.

Journal: Journal of immunological methods

Volume: 406

Pages: 143-147

eISSN: 1872-7905

ISSN: 0022-1759

DOI: 10.1016/j.jim.2014.03.010

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.

Source: Europe PubMed Central