- Warm the protein buffer, target protein, and 10X TurboBiotinylation Mix to 25 oC in a water-bath.
- Set up TurboBiotinylation Reaction
- Target protein at 1-5 mg/ml. Adjust volume with protein buffer.
- 1X TurboBiotinylation Mix
- 1X TurboBiotinylase DT
- Mix gently and incubate at 25 oC in a water-bath without agitation for 2 hours. Do not dialyze the reaction.
- TurboBiotinylase DT can be removed by GSH or Ni-IMAC affinity capture through its dual GST- and His-tags.
- Biotin and ATP can be easily removed by desalting columns or other columns. Multiple rounds of dialysis are needed to completely remove biotin and ATP.
Optimization of TurboBiotinylation Reaction:
The efficiency of biotinylation of a target protein depends on the stability of the target protein. Complete biotinylation is usually achieved in a buffer preferred by the target protein. In addition, an optimal biotinylation reaction requires sufficient concentrations of substrate and enzyme. TurboBiotinylation reaction at 25 oC with target protein of 40 µM and 1X TurboBiotinylase DT of 1.6 µM (0.1 mg/ml) leads to complete biotinylation of target proteins of different sizes (10-130 kDa) in most cases.
At 25 oC, many target proteins only need 0.05X TurboBiotinylase DT (5 µg/ml) to reach complete biotinylation within 1 hour.
If a target protein is not stable or aggregates at 25 oC or higher, TurboBiotinylation reaction can be carried out overnight at 4 oC. TurboBiotinylase DT has lower activity at 4 oC than at 25 oC. 5X-10X TurboBiotinylase DT may be needed for complete biotinylation of a target protein at 4 oC.
When target protein concentration is below 40 µM (1 mg/ml for a 25 kDa protein or 4 mg/ml for a 100 kDa protein), the biotinylation efficiency can be improved by increasing TurboBiotinylase DT in the reaction. Usually 5X TurboBiotinylase DT is sufficient to compensate for the low concentration of a target protein. For large target proteins (MW>100 kDa) at low concentration, it is recommended to use 5X TurboBiotinylase DT in a biotinylation reaction at 25 oC and 10X TurboBiotinylase DT in a reaction at 4 oC.
High concentrations of substrate and enzyme increase biotinylation efficiency. If a target protein is stable, a high concentration of the target protein is recommended. The rule of thumb is to use 1X TurboBiotinylase DT for 1 mg/ml target protein, 2X TurboBiotinylase DT for 2 mg/ml target protein, and so on.