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Biotin Labeling for Avitag Protein

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

价格 ￥1,100.00 （ 供应商现货 ： 3-5个工作日）
货号 UA070015
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产品介绍

产品介绍 评论(0)

产品规格

  • 分子别名

    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

  • Accession

    P06709

  • 表达序列

    Met1-Lys321

  • 表达宿主

    E.coli

  • 分子量

    35kDa (Reducing)

  • 纯度

    ＞95% by SDS-PAGE and HPLC

  • 内毒素含量

    <0.5EU/μg

  • 标记

    Unconjugated

  • 标签

    No Tag

  • 性状

    Liquid

  • 缓冲体系

    20mM Bicine, 50% Glycerol, pH8.3

  • 储存条件

    • 12 months from date of receipt, -20℃ as supplied.
    • Store BirA and BirA Buffer at -20℃.
    • Avoid repeated freezing and thawing cycles.

  • 文献引用

    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.

背景介绍

  • 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.

产品组分


操作步骤

  • 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.

  • 生物活性

    • 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.

  • 电泳

    • 2μg（N: non-reducing condition, R: reducing condition).

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