protein lysates preparation - (Aug/28/2004 )

Hi!
Could anyone please help me with my problem? I have a problem with preparing protein lysates for SDS page. I am using regular 2x sample buffer, which I mix 1:1 with cell suspension in PBS. Final concentration of cells in lysate is 10 milions cells/ml, but I get greasy substance, which I am not able to load into gel. I even tryed to decrease amount of cells but the result is the same. Is there a problem with some of the compounds of sample buffer? Or where could be a problem? Thank you

Hey-
I think that your "greasy" substance is really chromasomal DNA. It clumps up as a ball on the tip so you cant aspirate the lysate into the tip, right?
If so, I simply boil the sample as I would before loading, then vortex on high for about 30 seconds. This shears the DNA so it won't clump. Then spin the tubes to pellet any debris and load the supernatant.

Hope that helps

-or you could sonicate for 5s
-or add DNase to your buffer

Hi, thank you very much for both your answers. I will definitelly try it!

If normal boiling as for gel loading isn't sufficient, (much) longer boiling will do the job. I had to boil a sample for 30 minutes once before it was "pippetable"!

mike