ASGSB 1998 Annual Meeting Abstracts

[28]
BLOCKING OF BIOTINYLATED PROTEINS PRIOR TO USE IN THE AMPLIFIED ALKALINE PHOSPHATASE IMMUN-BLOT DETECTION. M.J. Palm and B. Johnson-Wint. Department of Biological Sciences, Northern Illinois University DeKalb, Il

The analysis of protein from tissue samples using a biotin-streptavidin detection system is common place in today’s cell and developmental biology lab. One analysis that uses this detection methodology is western blot detection using the Bio-Rad Amplified Alkaline Phosphatase Immun-Blot Detection. This detection format uses a biotinylated secondary antibody with the amplification of streptavidin. Then biotinylated alkaline phosphatase completes the sandwich and color developing reagent, 5-bromo-4-chloro-indoyl phosphate and nitroblue tetrazolium, is added. This forms a purple precipitate at any point of binding of the biotinylated alkaline phosphatase. In analyzing total protein pellets using extremely sensitive biotin-streptavidin detection, endogenous biotinylated proteins can give erroneous results through cross-reaction with the streptavidin in the detection kit. The purification of proteins, to eliminate this cross-reaction, can be labor intensive and time consuming therefore any reduction in time and cost of deleting nonspecific interactions is beneficial. In the model system when total protein extracts from bone are run on the western blot detection numerous non-specific bands are encountered. We have developed a methodology to block these endogenous biotinylated proteins from being detected in the western blot. The blocking step involves the use of avidin and biotin in a pre-primary antibody step to block the biotin-streptavidin interaction, this new step is called the BLOCKO step. The addition of this step allows the specific bands of interest to be viewed without elaborate purification of the total protein. The BLOCKO step is easy, cost-effective, and requires little time. This new information about detection of biotinylated proteins could also be used to identify endogenously biotinylated proteins from total protein extracts.

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