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.
Coomassie blue of protein gel shows a same result
Could anyone know what happened?
-stephenff-
QUOTE (stephenff @ Jul 11 2008, 02:02 PM)
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.
Coomassie blue of protein gel shows a same result
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.
Coomassie blue of protein gel shows a same result
Could anyone know what happened?
Never heard of this one. Transfer may be a culprit, but your coomassie blue negates that.
Did you do it twice? May be some problem with the sds-page? Does your marker lane come out fine? In that case, it wouldn't be a problem with gel.
Let us know what was the matter once you have tracked it down.
..
-cellcounter-
Confirm please that you cooled during bead lysis.
-jorge1907-