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작성일 : 18-01-27
[Accelagen] Biotinylation Kit for high efficiency in vitro biotinylation

 

TurboBiotinylationTM Kit

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

10% glycerol.

 

 

Protein Type:

Biotin ligase

 

Source:

E. coli

 

Specific Activity:

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 Purity:

 

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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.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TurboBiotinylase Activity:

 

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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.

 

 

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. Affinity purification can separate the biotinylated tagged target proteins from TurboBiotinylase, 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 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.

 

 

Ordering information

    

Catalog No.

Product Name

Size

B0114P

TurboBiotinylationTM Kit

50X TurboBiotinylase (0.05 mg)

10X TurboBiotinylation Mix (1 ml)

B0114M

50X TurboBiotinylase (0.5 mg)

10X TurboBiotinylation Mix (1 ml)

B0114L

50X TurboBiotinylase (2.5 mg)

10X TurboBiotinylation Mix (5 ml)

* Concentration: 2.5 mg/ml

 

 

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