I introduced a heterologous gene using pPIC 9 plasmid into KM71 strain and confirmed the presence of the target gene by PCR. The colonies (6) I am checking growing well on Minimal growth media (glycerol 2%) while control strain does not show any growth on MG media. The protein expected to be in the media, however, I can see it neither in the media (even concentrated) nor in cell lysate (1X loading buffer, boiled 95 C for 10 min) when performed SDS-PAGE followed by gel staining/western blot.
I also induced at 15 C with 0.5% MeOH (to reduce possible degradation by proteases) and carried out dot blot (1 to 25-50X concentrated) analysis for all 6 colonies at 24, 48 and 72 hours and still not seeing any clear dots (positive control working perfect, though). Tried 1% MeOH induction, yeastern blot as well and they also did not work.
Do you have a fluorescent antibody against your protein? Maybe you can check on microscopy if there is no expression at all, and after that decide what to do.
"(1X loading buffer, boiled 95 C for 10 min) when performed SDS-PAGE followed by gel staining/western blot". 10 min is too long; 4-5 minutes will do.
Dear Chandra Somasundaram ,
Thank you for your point but does keeping the cells may degrade the proteins in the conditions mentioned above?
Yes. why did you boil the cell lysate for 10 minutes? it should not be over 5 minutes.
I used to incubate for five minutes with E.coli but I heard that yeast cells are harder to break down so I increased the incubation period.
Muzaffar Muminov sometimes i have had problems with boiling samples, you may sonicate your cells to lyse them, it may give better results sometimes if you are doubting about your lysis efficiency.
However, you will need a way to check whether your protein is expressed in any moment during yeast growth, not just at the end of culture.
Today I have finally seen it with a very concentrated sample. Seems I need to screen for better producents.
Thank you for everyone.
Muzaffar Muminov
There may be problem with lysate buffer. If you didn't detect any desired protein. Can you sonicate the cells to get more yield !!
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