Quality control assay: Biotin acceptor peptide tag fusion protein was used as a substrate. BirA activity was verified by streptavidin binding and mass spec analysis. A >95% bitinylation of the substrate was confirmed under the experimental conditions.
Protocol for biotinylation
1. BirA reaction condition: 50mM bicine buffer, pH 8.3, 10mM ATP, 10mM MgCl2, 50μM d-biotin.
2. Substrate concentration should be around 0.5-1mg/mL. Add 10-50 ug birA per mL reaction volume.
3. Incubate at room temperature for 1 hour or more. Longer reaction time is recommended at lower temperature.
|Product Name：||Biotin ligase BirA|
|Application：||FOR LABORATORY RESEARCH USE ONLY.|
|Formulation：||20 mM MES buffer at pH5.5, 50 mM NaCl, 10% glycerol.|
|Purity：||80% by SDS-PAGE gel|
|Storage：||Long term storage at 80 C. Avoid repeated freeze-thaw cycles.|
|Note：||Biotin/biotinylated proteins are more soluble in bicine buffer, therefore it is recommended. High glycerol and NaCl conc. may lower birA acitivity.|