Met1-Lys321
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK.
35kDa (Reducing)
＞95% by SDS-PAGE and HPLC
1.Howarth M, Ting AY. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat Protoc. 2008;3(3):534-45.
2.Fairhead M, Howarth M. Site-specific biotinylation of purified proteins using BirA. Methods Mol Biol. 2015;1266:171-84.
BirA, the most characterized biotin-acetyl-CoA-carboxylase ligase, has been successfully applied to biotin labeling with a wide range of protein substrates that fused an acceptor peptide (AP, also known as Avitag) in vivo and in vitro. In the presence of ATP, BirA activates biotin to form the BirA-biotinyl-5'-adenylate (BirA-bio-5'-AMP or holoBirA) complex. BirA can either transfer the biotinyl moiety to the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. Due to its extremely high strength, the interaction between biotin and streptavidin has been exploited for biotechnological applications, like in vivo imaging, cell sorting, et al.
Recommendation before Ligation
1. Higher protein concentration (above 40μM) is always recommended to be biotin labelled. It takes more time to label target protein, if protein concentration is lower.
2. 2.0μg BirA is recommended to ligate D-biotin to 10nmol of Avi tagged protein at 40μM in 30-60 min at 37℃ or 60-90min at RT. Ligation reaction can also be performed at 4℃, but much more time required, like overnight.
3. ATP solution is vulnerable to hydrolyze with time, so longer incubation time may be problematic the target protein from ATP, lowering the ligation efficiency.
4. 10X Buffer is provided
Add the following to a microcentrifuge tube:
| Items | Volum |
|
Avi tagged Protein |
10nmol |
|
10X Buffer A |
25μl |
|
10X Buffer B |
25μl |
|
10X Buffer C |
25μl (If necessary)* |
|
BirA |
2.0μg |
|
ddH2O |
To 250μl |
*When the concertration of substrate between 40-80μM,We recommend our customers add Buffer C
5. Mix and incubate at 37°C. Take 20μl aliquots at 30min, 60min, 90min to analysis.
6. Analyze by Pulldown or other Biotin quantitative kit.
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