What does this mean if Western blot analysis with anti-CBD serum detects a 55 kDa product (when the C-terminal TYB fusion vectors are used) and not the fusion precursor?
This data indicates that either the target protein is degraded or cleaved in vivo. The protein samples should be prepared in SDS-PAGE sample buffer without DTT (or 2-mercaptoethanol) since boiling in DTT-containing sample buffer may cause cleavage of the fusion protein. If possible, perform a Western blot with target protein antiserum to differentiate between proteolysis versus intein-mediated cleavage. If proteolysis is evident, try different hosts. It is believed that in vivocleavage is caused by hydrolysis of the thioester linkage between the target protein and the intein. In vivocleavage may be reduced by inducing cells at lower temperatures (for example, 12-15°C overnight or room temperature for 3-6 hours). Another possiblity is to include a favorable residue or sequence immediately adjacent to the cleavage site which may improve controllable cleavage. Use of a pTXB vector may reduce in vivo cleavage and increase the final yield. Furthermore, expression using a N-terminal fusion v
Related Questions
- What does this mean if Western blot analysis with anti-CBD serum detects a 55 kDa product (when the C-terminal TYB fusion vectors are used) and not the fusion precursor?
- What does Product, Market, Competitive and Financial Data Acquisition and Analysis mean?
- What does "enriched" mean with a Western blot test?
