Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.
Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.
Banks
Rosamonde E
RE
Cancer Research UK, Clinical Cancer Centre, St James's University Hospital, Leeds, UK. r.banks@leeds.ac.uk
Craven
Rachel A
RA
Harnden
Patricia A
PA
Selby
Peter J
PJ
D001710:Biotin / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods*; D007668:Kidney / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000378:metabolism*
D011506:Proteins; D001710:Biotin; D019809:Streptavidin