Total cellular protein from lysate? - (Jun/18/2007 )

Hi all,

I try to do WB on total cellular protein from HEK 293 cells. But after 10' lysis in 1%SDS/10%glycerol/PBS at RT and taking out from -80˚C, the samples contain jelly-like components which is too viscous to pipet. I guess that is cell debris such as membrane and nucleus. Do you have this problem and maybe I should try other lysis buffer?

Thanks

Usually that is the DNA you can make the solution less viscous by degrading the DNA (usually accomplished by something like sonication) then you will be able to work with your sample.

HTH

Try adding DNase 1 (1ug/100ml) to the lysis buffer.

Are you centrifuging the samples after lysis? I centrigue for 15 min at 13,000rpm and then take only the supernatant for western blot.

Thank you guys.

I centrifuged 10' at 14000rpm after taking sample out from -80 the day I performed WB, but it usually doesn't work and very hard to separate the jelly-like things from supernatant. I don't samples in hand right now and guess will try DNase next time.

Try to boil the sample for 10 minutes, it will no longer be viscous.

This raise another question, what cause DNA degradation during the boiling?
Dose DNA alone cause the viscosity? Or the large DNA/proteins complex was responsible for that?
So boiling may reduce the viscosity by dissociating the complex without destroying the DNA.