Protein expression Using NEB yest kit - (Apr/04/2007 )
I have cloned one gene of around 0.65 kd in a pKLAC1 yeastvector and transformed in K. Lactis (From NEB). After successful transformation i tried to express the protein but i cannot seee the protein in the gel. Although the control construct expressed very well. Then i thought might be the low expression making it difficult to see in the SDS PAGE gel. Then i precipitated the protein by ammonium sulphate 100ml then bring it to 1 ml finally. After runing the gel with this concentrate even i cannot see any thing. Has anybody familiar with this kit? please give me some suggestion?My protein expression is expected to come in medium(secreted protein)
what are your sds-page conditions (buffer system, acrylamide %, etc.).
your protein is very small. you will have to use the tris-tricine buffer system with a high acrylamide% gel in order to visualize it.
you may also have to change your staining method based on the amino acid content.