Biotinylation Kit for high efficiency in vitro biotinylation
Accelagen's TurboBiotinylationTM Kit is an enhanced biotinylation kit for in vitro biotinylation of biotin-
accepting proteins and recombinant proteins with AviTag TM or 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
TurboBiotinylase has a specific activity of >8,000 Units/µg.
One unit of TurboBiotinylase 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 at -80℃ and 10X TurboBiotinylation Mix at 4℃.
Components and Formulation:
1.50X TurboBiotinylase at 2.5 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 is a stable ultra-pure recombinant E. coli BirA
biotin ligase purified from E. coli and has a calculated molecular
weight of 35.6 kDa. It is free of protease or nuclease contamination.
The efficiency of biotinylation can be measured by mass
spectrometry. Biotinylation adds 226.31 Da to a protein.
Alternatively, biotinylation can be evaluated by streptavidin-agarose
pull-down. A Coomassie stained gel will visually show the
efficiency of biotinylation.
1. Remove biotin and ATP from the biotinylation reaction sample by
a spin desalting column.
2. Save sample as Total (T)
3. Incubate with streptavidin-agarose for 1 hour with agitation.
4. Spin down the streptavidin-agarose.
5. Save the supernatant as Unbound (U)
6. Save the streptavidin-agarose as Bound (B)
7. Run gel and stain with Coomassie Blue.
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. Affinity purification can separate the biotinylated tagged target proteins from TurboBiotinylase, biotin,
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 of 1.6 µM (0.05 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 (2.5 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 has lower activity at 4℃ than at 25℃. 5X-10X TurboBiotinylase 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 in the reaction. Usually
5X TurboBiotinylase 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 in a biotinylation
reaction at 25℃ and 10X TurboBiotinylase 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 for
1 mg/ml target protein, 2X TurboBiotinylase for 2 mg/ml target protein, and so on.