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?
If it is not proteolysis, then it may be something in the lysate that is preventing transfer to NC. Look at the proteins on coomassie stained gel to see how it compares to the NC staining. Or add MW standards (unstained) to a lane with lysate, and see how they transfer. Also maybe compare lysate with cells boiled in loading buffer. Maybe you are vortexing the cells too long with the beads and sheairng proteins (not sure if that can happen).
Thanks for help. I did tried coomassie staining of the gel, but the result appeared similar as ponceau red staining.
As for lysis time, I also tried shorter time.
You didn't mention cooling - you could be denaturing the protein if the temp of bead beating gets too high
But surely denaturing is simply that - denaturing of the 3-d structure as opposed to breaking covalent bonds. It would have to be high and lengthy heating to actually break the covalently bound peptide bonds to produce the smaller fragments, right?
What protease inhibitors were you using ?