Avi标签蛋白生物素标记试剂盒; 生物素标记试剂盒，生物素标记，标记， Bifunctional ligase/repressor; BirA; Biotin-protein ligase
形态: 20mM Bicine, 50% Glycerol, pH8.3;
分子量: 35 kDa (Reducing);
纯度: ＞95% by SDS-PAGE and HPLC;
|BirA||20mM Bicine, 50%Glycerol, pH8.3||200ug||1mg|
|10×Buffer A||500mM Bicine, pH8.3||2.5mL||12.5mL|
|10×Buffer B||100mM ATP, 100mM MgOAc, 500uM D-Biotin||2.5mL||12.5mL|
|10×Buffer C||500uM D-Biotin||2.5mL||12.5mL|
Recommendation before Ligation
1. Higher protein concentration (above 40uM) is always recommended to be biotin labelled. It takes more time to label target protein, if protein concentration is lower.
2. 2.0ug BirA is recommended to ligate D-biotin to 10nmol of Avi tagged protein at 40uM 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. 10×Buffer is provided
Add the following to a microcentrifuge tube:
|Avi tagged Protein||10nmol|
|10×Buffer C||25uL (If necessary)*|
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.
12 months from date of receipt, -20℃ as supplied. Store BirA and BirA Buffer at -20℃. Avoid repeated freeze-thaw cycles.
1.Howarth M, Ting AY. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat Protoc. 2008;3(3):534-45. doi: 10.1038/nprot.2008.20. PMID: 18323822; PMCID: PMC2671200.2.Fairhead M, Howarth M. Site-specific biotinylation of purified proteins using BirA. Methods Mol Biol. 2015;1266:171-84. doi: 10.1007/978-1-4939-2272-7_12. PMID: 25560075; PMCID: PMC4304673.
Note: 10nmol Avi-tagged protein substrate were labeled by 2μg BirA at 37℃ for 30min. All sample lanes are loaded 4μg protein, respectively. Streptavidin has 4 biotin-binding sites, and may associate with 1 to 4 chains of the biotinylated protein. Therefore, the extent of protein biotinylation can be calculated based on the ratio of biotinylated protein to original substrate protein.