The BIS-300 Kit contains a fully biotinylated MBP-AviTagTM fusion protein standard and an unbiotinylated MBP-AviTagTM fusion protein that may be used as an "extent-of-biotinylation" comparison for BirA biotin-protein ligase reactions, for SDS-PAGE gel analysis or in Western blot analysis.
Kit supplied with the following:
• One (1) vial containing 50μL of fully biotinylated MBP-AviTagTM fusion protein at 1mg/mL (50μg).
• One (1) vial containing 100μL of unbiotinylated MBP-AviTagTM fusion protein at 1mg/mL (100μg).
By comparing a known amount of a 100% biotinylated AviTagTM fusion protein as a standard against a known amount of BirA-reacted protein-of-interest in an ELISA-type, multi-well dilution format (called BRTA for Biotinylation Reaction Titration Assay) it is possible to assess the extent of biotinylation achieved in your own BirA biotinylation reactions in a highly accurate and reproducible manner.
In addition, a mass shift between biotinylated and unbiotinylated fusion proteins visible on both SDS-PAGE and Western blot can be used to subjectively determine the amount of biotinylation that has occurred in a reaction by the observation of differences in band density in a mixed population of biotinylated vs. nonbiotinylated substrate.
The BIS-300 Kit contains the fully biotinylated standard used for the BRTA protocol and an unbiotinylated AviTag’d protein to react in a parallel to your BirA/protein biotinylation reaction as a positive control. A link to the BRTA protocol can be found here.
