As shown below, biotinylation can be done with using biotin ligase and biotin with a BAP tagged fusion protein.
- Synthesize target protein with BAP tag by 2nd PCR
- Transcript product of #1 and synthesize mRNA
- Prepare BirA, biotin ligase, and Biotin
BirA can be easily found in E. coli. So you can get the gene with following 1st primer. Also our kit can synthesize it as In vitro.
- Follow translation solution mentioned below.
- After translation, to remove biotin, dialyze the solution with appropriate buffer.
- Please note the following for detection and purification
membrane blocking with protein is not possible.
Denatured elution due to strong binding with biotin force