Sonicating cell lysate for Westerns - (Jun/05/2005 )

Hi Guys,
I have some very silly and for some of the experts...stupid questions. But my western woes dont seem to be ending

My protein cell lysates seems to be fine after I sonicate it in cyclin ripa. However when I load it on to the gel there seems to be some of the stuff still stuck on the gel well and does not migrate. therfore,

1. How long are you supposed to sonicate a 75 ul sample??
2. Should the microtip touch the pellet?? Some tell me that the tip should be just at the top of the liquid and should get submerged.
3. Some tell me that once the sample turns "milky" my sonication is done.

I do the sonication in the presence of all the protease inhibitors etc and under ice. I make sure that I place my tip also under ice for 20 mins before starting. so..what am i doing wrong?

Any help will be appreciated.
Thanks a lot
p..

hi
protocol from my colleague
Pellet cells (200g 5' to 10')
discard supernatant.
Vortex at gently speed but maintain until the pellet is well broken (like a milky solution, not a good comparison but i suppose it's enough clear)
chill on ice for 2' (less if your pellet becomes frozen but at least 30'')
sonicate 4 times of 30'' each, separated by 30'' in ice.
Centrifigation 20 000g for 30' in order to discard the "detritus" (i know it's not the correct word so please may you correct me?)
Ready for quantitation and use.


if you try it, please tell us the result.

good luck
Fred

hi Fred,
Thanks a lot. will give it a try.

Since you gave me the liberty to correct you I think you meant "Debris"?

But got the message. Thanks again.
Regards..
p...