Having problem preparing sample for 2D gel. Help pls. - (Feb/01/2006 )

Hi guys,
I am trying to prepare total protein sample from Cortical neron lysate. What I did is that I lyse the cells in lysis buffer containing 1% NP-40 and Deoxycholate for 30 min on ice, centrifuge and take the supernatant. Then I did TCA percipitation followed by acetone wash multiple times. The pellet was air dried. My problem is the result pellet from the above treatment was not soluble in the bio-rad rehydration buffer. The property of the pellet is like some kind of gel and it is somehow sticky. Does anyone have the same problem? how do your prepare your sample from a total lysate?
Thank you very much.

Tianshu

QUOTE (Tianshu @ Feb 1 2006, 03:52 PM)

Hi guys,
I am trying to prepare total protein sample from Cortical neron lysate. What I did is that I lyse the cells in lysis buffer containing 1% NP-40 and Deoxycholate for 30 min on ice, centrifuge and take the supernatant. Then I did TCA percipitation followed by acetone wash multiple times. The pellet was air dried. My problem is the result pellet from the above treatment was not soluble in the bio-rad rehydration buffer. The property of the pellet is like some kind of gel and it is somehow sticky. Does anyone have the same problem? how do your prepare your sample from a total lysate?
Thank you very much.

Tianshu

the "gel like" pellet is due to too much detergent don't use so much detergent or even try to use sonication to break the cells .

I hope it will work