Hi, 

I am sure you have check it but do you have any control of your anti-body? One you have done this you have to discard a false positive. Have you check by PCR that  your gene has been efficiently introduced? When you confirm that your gene is there and the antibody is working there is something else you can do, instead of using sucrose you can use raffinose before galactose induction. Using raffinose you will have a stronger GAL response. Therefore if you are accumulating low amount of your protein, maybe this will alow you to have higher. 

If you have a problem of premature protein degradation, before using proteasome inhibitors (that as you may know are really expensive) try to do a time course at different temperatures, some times reducing the temperature is good. 

However, if after all these things you still have problems with the induction I suggest you to check two things, after long induction how cell looks like? And second, do serial dilutions in YPGal plates to see if the expression is somehow toxic. 

Hi,

It seems that there is recombinant protein present in your sample (activity on plate), but you just can't detect the protein by SDS-PAGE and Western blot. In the case of SDS-PAGE, which staining method do you use? If the amount of protein is really low, it is best to use a silverstain (or equivalent) rather than Coomassie Blue, as the former detects much lower amounts of protein than the latter (but it is more expensive). Secondly, how sure are you about the antibody that you use for Western blot? In other words, did you include a positive control, which allows you to conclude that the assay worked perfectly? If there is nothing wrong with the Western blot method, I think you might be dealing with protein degradation, as mentioned in earlier posts. In that case, you might add casamino acids to the culture medium during induction to deal with protease activity. Also lowering the temperature during induction and the induction time might reduce protein degradation.

Thanks for prompt response. The western blot is clean all the way and did not notice any spot. My protein supposedly present on the surface of EBY100 yeast strain covalently joined by Aga2 anchoring region. Hey Roger, I have confirmed gene insertion in vector by PCR before galactose induction. I am wondering if raffinose could make GAL promoter efficient. I will definitely try, however I appreciate if forward relevant paper in this regard. Regarding toxicity I have not checked this before