TurboBiotinylation DT^TM Kit

1. Warm the protein buffer, target protein, and 10X TurboBiotinylation Mix to 25 ^oC in a water-bath.
2. Set up TurboBiotinylation Reaction
   • Target protein at 1-5 mg/ml. Adjust volume with protein buffer.
   • 1X TurboBiotinylation Mix
   • 1X TurboBiotinylase DT
3. Mix gently and incubate at 25 ^oC in a water-bath without agitation for 2 hours. Do not dialyze the reaction.
4. TurboBiotinylase DT can be removed by GSH or Ni-IMAC affinity capture through its dual GST- and His-tags.
5. 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.

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.