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CS0008

Sigma-Aldrich

Enzymatic Protein Biotinylation Kit

for biotinylation of up to 20 mg target protein

Synonym(s):

Biotin labeling kit

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About This Item

UNSPSC Code:
12161503
NACRES:
NA.84
Pricing and availability is not currently available.

Quality Level

200

shipped in

dry ice

storage temp.

−70°C

General description

Biotinylated proteins are used in a variety of applications, such as binding assays, biopanning, and protein purification. Although chemical biotinylation methods are common, they present several challenges, including partial labeling and high batch to-batch variation, because of the random nature of these methods. In addition, the lack of control of the biotinylation site might affect the biotinylated protein′s activity. The Enzymatic Protein Biotinylation Kit allows direct and specific biotinylation using a specific biotin ligation enzyme. The kit contains all the components required for in vitro biotinylation of up to 20 mg recombinant target protein. Biotinylation is mediated by the enzyme BirA biotin ligase, which uniformly ligates biotin to lysine residues in the context of several tags, such as AviTag™ and Biotag. This kit was tested successfully under a wide range of conditions, including:
  • Temperatures ranging from 4 °C to 30 °C
  • pH from 6.0 to 9.0
  • NaCl concentrations from 50 to 500 mM.
The kit includes streptavidin, which can be used to assess the degree of biotinylation. The kit also includes a BLPR-tagged protein as a positive control.
Supportive calculator (Click here to download a calculator excel file): Will calculate the reagents needed according to your experimental needs.

Legal Information

AviTag is a trademark of Avidity, LLC

Storage Class Code

10 - Combustible liquids

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Certificates of Analysis (COA)

Lot/Batch Number
0000417698
0000417697
0000417697
0000417698
0000364045

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Questions

1–2 of 2 Questions  

  1. The gel for product #CS0008 suggests a similar activity of the kit at different pH values (6 - 9). However, in my hands, the reaction proceeds significantly faster at pH 8.3 compared to pH 6/7. Which conditions were used for the shown labeling reactions?

    1 answer

    1. The labeling reactions for this kit were performed at pH 7.

      Helpful?

  2. Is the BirA protein tagged in any way (ideally FLAG) in order to remove it from the reaction mixture?

    1 answer

    1. The BirA ligase is not tagged. There is no suggested protocol for the removal of excess ligase. However, in the presence of ATP and biotin the reaction will run to completion. Any residual ligase will remain inert. The BirA ligase has a molecular weight of 33.5 kDa. Note that excess biotin is removed by dialysis. Please see the link below to review the kit Technical Bulletin:
      https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/700/170/cs0008bul-ms.pdf

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