• Substrate peptide (protein) containing AviTag™ (for concentration see discussion below)
• BIO200 as necessary (see note below).
To ensure a rapid rate of biotinylation, it is recommended that the AviTag’d substrate be as close as
possible to 40μM in the final reaction mix. The lower the substrate concentration in the reaction mix,
the longer it will take for biotinylation to complete. For example, a substrate at 40μM may be
completely biotinylated within 30-40 minutes, but at 4μM it will take more than 5 hours using the same
amount of BirA enzyme. To complete the biotinylation reaction at 4μM in under an hour (i.e. 10 times
faster), it is necessary to add 10 times more enzyme to the reaction mix. As it is, the amount of BirA
enzyme added to the reaction mix may need to be varied to achieve biotinylation within an acceptable
time-frame (see below).
The BirA enzyme has been tested ａnd shown to be free from measurable protease contamination.
Longer incubation times do carry the risk that your target protein may be proteolyzed from proteases
present in small amounts from target protein purification, ａnd we recommend using the shortest
incubation time that fully biotinylates the target protein. Longer incubation times may be alos
problematic the target protein from ATP. ATP in solution will hydrolyze with time, lowering the available
ATP for the biotinylation reaction. Therefore completing the reaction quickly will be beneficial to
achieving maximal biotinylation.
Biomix B contains ATP that might subject to hydrolysis if not handled correctly. Biomix B is the most
labile component of the BirA reaction kit, ａnd the Biomix B should be thawed on ice ａnd immediately refrozen
after addition to the BirA reaction.
Typically, for every 10 nmol of substrate (at 40μM), we recommend 2.5μg of BirA enzyme to complete
the biotinylation in 30 - 40 minutes at 30°C or about 1 hour at room temperature. Reactions can be
performed at 4°C but will require more time, overnight if it is convenient. This is acceptable because the
ATP hydrolyzes more slowly at 4°C. Also, additional ATP in solution may be added to the reaction if there
is a question of incomplete biotinylation due to ATP degradation. The other reactants are more than
stabile enough to continue the biotinylation if this is the case.
The BiomixB has been optimized for the biotinylation of substrates at concentrations of no more than
40μM. If it is desired to biotinylate substrates at concentrations of 40-80μM, then it is necessary to
supplement the reaction mix with additional biotin. The BIO200 (500μM d-biotin) has been included to
supplement reaction mixes of up to 80μM. For substrate concentrations above 80μM, please contact
our Technical Support at (720) 859 6111 or Toll-Free (877) 333 6063.
Example calculation for determining the amount of BirA needed for your reaction:
For this example we will assume we are using a peptide substrate with AviTag™ of 15 kDa MW (note:
the AviTag™ is 1.829 kDa MW).
1 nmol of a 15 kDa protein = 15μg; therefore 10 nmol = 150μg (2.5μg of BirA is recommended per 10
nmol).
For a 1mg/mL solution of the substrate, 0.5mL would contain 500μg of that substrate.
- 500μg substrate/(150μg substrate/10 nmol) = 33.3 nmol of substrate to be biotinylated
- 33.3 nmol of substrate/(10 nmol of substrate/2.5μg BirA) = 8.325μg BirA
For best efficiency the substrate needs to be at 40μM concentration. In our example we have currently
0.5mL at 1mg/mL.
- 1mg/mL = 1000μg/mL = 66.6 nmol/mL = 66.6 μmol/L = 66.6μM.
We need to dilute the reaction solution to 40μM final.
- (66.6μM/40μM)(X/0.5mL); X = 0.83mL
So the final reaction mix would need to be brought up to 0.83mL with BiomixA (10X), BiomixB (10X),
BirA ａnd water (or your buffer of choice, see precautions below).
- 500μL of 66.6μM substrate + 83μL BiomixA + 83μL BiomixB + (164μL – XμL of BirA for 8.325μg*)
water + XμL BirA = 0.83mL final reaction volume at 40 μM substrate concentration. (*BirA is
offered at 1mg/ml or 3mg/mL concentrations).
Examples of BirA quantity per substrate amount for three different
Substrate
MW
10 nmol
substrate
nmol of substrate in 0.5mL of
1mg/mL solution (500μg)
Amount of BirA
needed for 500μg*
Reaction volume
at 40μM
15 kDa 150 μg 33.3 nmol/500μg 8.325 μg 500μg/0.83 mL
75 kDa 750 μg 6.67 nmol/500μg 1.67 μg 500μg/0.17 mL
225 kDa 2250 μg 2.2 nmol/500μg 0.55 μg 500μg/0.06 mL
(*at 2.5μg BirA per 10 nmol AviTag’d substrate)
Note from the table above that it may be necessary to concentrate your substrate to meet the 40μM
criteria.
