Products

TurboBiotinylation DT^TM Kit

Biotinylation Kit for high efficiency in vitro biotinylation

Accelagen's TurboBiotinylation DM^TMKit is an enhanced biotinylation kit for in vitro biotinylation of biotin-

accepting proteins and recombinant proteins with AviTag ^TMor other biotin-accepting peptides. Combining

concentrated TurboBiotinylase and stable 10X TurboBiotinylation Mix, this kit offers high efficiency in vitro

biotinylation from 4-37℃.

It does not require any special buffer for its activity and is compatible with a wide range of buffers that are

most suitable for target proteins. This kit works equally well across pH 5.5-10, between 0-1 M NaCl, and in

10% glycerol. 

Protein Type:

Biotin ligase

Source:

E. coli

Specific Activity:

TurboBiotinylase DT has a specific activity of >4,000 Units/µg.

One unit of TurboBiotinylase DT completely biotinylates 1 pmol of a substrate protein (23 kDa) at

concentration of 1 mg/ml within 1 hour at 25℃.

Storage and Handling:

TurboBiotinylation Kit is shipped with gel packs.

The kit retains its full activity after incubation at 25℃ for over a week.

All components of the kit can be stored at 4℃ for at least 6 months. For long term storage, store 50X

TurboBiotinylase DT at -80℃ and 10X TurboBiotinylation Mix at 4℃.

Components and Formulation:

1. 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.

2. 10X TurboBiotinylation Mix (50 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 100 mM ATP, 5 mM biotin)

TurboBiotinylase Purity:

TurboBiotinylase Activity:

TurboBiotinylation Reaction:

1. Warm the protein buffer, target protein, and 10X TurboBiotinylation Mix to 25℃ 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

3. Mix gently and incubate at 25℃ 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.

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℃ 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℃, 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℃ or higher, TurboBiotinylation reaction can be carried out

overnight at 4℃. TurboBiotinylase DT has lower activity at 4℃ than at 25℃. 5X-10X TurboBiotinylase DT may

be needed for complete biotinylation of a target protein at 4℃.

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℃ and 10X TurboBiotinylase DT in a reaction at 4℃.

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.