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Problem with protein expression using Pichia Pastoris


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#1 vlitx

vlitx

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Posted 03 May 2010 - 08:00 PM

Hi..Im a master student that doing protein expression on PSMA using Pichia Pastoris.
The problem i have is the negative control that i do for the expression.
The BMGY medium,that containing glycerol were supposedly to allow the cell to grow. Than i will transfer it into BMMY that contain the methanol that will allow the protein expression.
At this point,i will keep one flask transfer to BMMY which i put glycerol instead of methanol,and supposedly it wont have any expression if i do western blot for that single flask because there is no methanol to induce the expression,juz the glycerol to keep the cell to grow.
But the problem is i keep getting the band for this negative control which mean there is a protein express and its already been for 4months.
The band that i get is better compare to the one that i growth in methanol and it is at the expected size.

Is there any one that know how to solve this?and is there any logical explanation? cause i have been trying for so many time,
and contamination of methanol in glycerol flask is not the problem.




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