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PRODUCT DETAIL
DATA SHEET
PROTOCOL
Components and Formulation:
50X TurboBiotinylase DT at 5 mg/ml (80 µM) in Storage Buffer of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM TCEP, 50% glycerol.
10X TurboBiotinylation Mix (50 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 100 mM ATP, 5 mM biotin)
TurboBiotinylase Purity:
TurboBiotinylase DT is a stable ultra-pure recombinant E. coli BirA biotin ligase with dual GST- and His-tags. It is purified from E. coli and has a calculated molecular weight of 62.4 kDa. It is free of protease or nuclease contamination. It can be easily removed by affinity capture through its GST- or His-tag.
TurboBiotinylase Activity:
The efficiency of biotinylation can be measured by mass spectrometry. Biotinylation adds 226.31 Da to a protein.
Alternatively, biotinylation can be evaluated by streptavidin-agarose pull-down. A Coomassie stained gel will visually show the efficiency of biotinylation.
Remove biotin and ATP from the biotinylation reaction sample by a spin desalting column.
Save sample as Total (T)
Incubate with streptavidin-agarose for 1 hour with agitation.