Biotin Labeling for Avitag Protein

Recommendation before Ligation

1. Higher protein concentration (above 40μM) is always recommended to be biotin labelled. It takes more time to label target protein, if protein concentration is lower.

2. 2.0μg BirA is recommended to ligate D-biotin to 10nmol of Avi tagged protein at 40μM in 30-60 min at 37℃ or 60-90min at RT. Ligation reaction can also be performed at 4℃, but much more time required, like overnight.

3. ATP solution is vulnerable to hydrolyze with time, so longer incubation time may be problematic the target protein from ATP, lowering the ligation efficiency.

4. 10X Buffer is provided

Add the following to a microcentrifuge tube:

*When the concertration of substrate between 40-80μM,We recommend our customers add Buffer C

5. Mix and incubate at 37°C. Take 20μl aliquots at 30min, 60min, 90min to analysis.

6. Analyze by Pulldown or other Biotin quantitative kit.