protein extraction from yeast--all extracted proteins are below 50KD - (Jul/11/2008 )
Hi, all. I grew yeast cells in galactose medium till stationary phase. Cells were harvested and suspended in PBS buffer. Cell lysis was done using glass beads. Before I move on to do Co-IP of one specific protein, I runned SDS-PAGE and nitrocellulose membrane transfer, followed by Ponceau Red staining. Strangely, all the visible protein bands are below 50KD.
I asked around and was told it might be due to incomplete lysis of cells. Then I increased the amount of glass beads and lysis time (I even tried 1minx5). But the result was still the same.
I used protease inhibitors in all the experiments.
Could anyone know what happened?
-stephenff-
QUOTE (stephenff @ Jul 11 2008, 06:46 AM)
Hi, all. I grew yeast cells in galactose medium till stationary phase. Cells were harvested and suspended in PBS buffer. Cell lysis was done using glass beads. Before I move on to do Co-IP of one specific protein, I runned SDS-PAGE and nitrocellulose membrane transfer, followed by Ponceau Red staining. Strangely, all the visible protein bands are below 50KD.
I asked around and was told it might be due to incomplete lysis of cells. Then I increased the amount of glass beads and lysis time (I even tried 1minx5). But the result was still the same.
I used protease inhibitors in all the experiments.
Could anyone know what happened?
I asked around and was told it might be due to incomplete lysis of cells. Then I increased the amount of glass beads and lysis time (I even tried 1minx5). But the result was still the same.
I used protease inhibitors in all the experiments.
Could anyone know what happened?
if you feel that proteolysis is not the main reason (degradation looks very characteristic beside absence of larger polypeptides), I suppose insufficient blotting performance (too short time, wrong transfer buffer etc.) try tank blotting to transfer larger polypeptides, too
-The Bearer-