No expression seen on PAGE - Shall I still go for costly protein-activity assay? (Jan/07/2009 )
I am tring to express a plant gene in E. coli Top10 strain. After protein extraction, the SDS-PAGE profiles of induced positive culture (having insert gene of my interest) and the negative control (induced but not having inset-only vector) appear similar. There is a band at expected size, but it is present in both of the above cases with the similar intensity. Now, to assay enzyme acivity is a costly affair for me because of the expensive substrates. My question now is shall I go for assaying ($$) the crude extract considering that there might be a very low expression not reflected in PAGE? Has anybody got expected protein acivity without seeing differential protein profiles in test and control samples?
can you immunoblot?
if not then you may have to bite the bullet and assay for the protein.