Biotin is frequently used in biochemical research because of it highly selective and very strong binding to Streptavidin or Avidin. This interaction is also used to conveniently immobilize proteins for purification, detection of molecular interactions for example using methods like surface plasmon resonance (SPR), or to be applied in functional assays.
This service will prepare and provide the biotin-labelled proteins using enzymatic biotinylation method that utilizes E. coli biotin ligase BirA on the wheat germ cell-free expression system.
While proteins can be biotinylated under different conditions, one elegant method uses the BirA biotin ligase for enzymatic modification of proteins having a matching biotin ligation site (bls) comprising 15 amino acids. This short sequence can be added to target proteins for obtaining afterwards mono-biotinylated proteins carrying only one biotin moisture at a defined position, a lysine within the recognition sequence. This is preferable to avoid any effect of the biotin moisture on the protein modification on functions of the target protein as principally possible when using other methods for random biotinylation.
Working with our wheat germ cell-free protein expression system, the BirA ligase can be directly added to the translation reaction to directly obtain a biotinylated protein at the end of the protein expression reaction. Depending on the indented use of the biotinylated protein, customers may work with crude reaction mixtures containing other proteins from the wheat germ extract, or purified protein commonly obtained by working with an affinity tag. In our experience, the BirA ligase does not biotinylated proteins within the wheat germ extract thus yielding in background free protein modifications have been directly used for example on the PerkinElmer AlphaScreen platform. You can find several examples for such studies in our reference list.
Service contents
Protein expression and biotinylation test
✓ Protein expression test
✓ Biotinylated protein expression
✓ Affinity purification e.g. His-tag or GST-tag *
✓ Biotinylation efficiency measured using the HABA method *
*We do not carry out HABA test when providing crude reaction mixtures. Those contain other proteins from the wheat germ extract that interfere with the HABA assay.
Note: This service does not guarantee protein yields and biotinylation levels.
Delivery time: 4 Weeks~
Flow of service
Scaling up biotinylated protein expression for production
✓ Scaling up biotinylated protein expression
✓ Affinity purification e.g. His-tag or GST-tag *
✓ Biotinylation efficiency measured using the HABA method *
*We do not be carry out HABA test when providingcrude reaction mixtures. Those contain other proteins from the wheat germ extract that interfere with the HABA assay.
Delivery time: 4 Weeks~
Flow of service
FAQ
How is biotinylation efficiency? |
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In the case of purified biotinylation dihydrofolate reductase (DHFR) [0.3 mg/ml], the biotinylation efficiency is 90%. |
How do you measure biotinylation efficiency? |
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Biotinylation efficiency measured using the HABA method. |
Inquiry
Feel free to contact us for any question, please contact below.
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