cell lysate problem

Grant Stewart g.s.stewart at bham.ac.uk
Sun Jul 19 12:51:37 EST 1998


When I am preparing samples for Western I do the following:

Thaw pellet of gamma-radiation treated LCLs (~4,000,000 cells) at 37oC.
Resuspend pellet in 200ul UTB buffer (9M urea, 150mM B-mercaptoethanol,
50mM Tris pH7.5). Sonicate for 2x15 seconds, keeping cool, then centrifuge
for 25mins at 35000 rpm at 10oC. 

The problem is that after centrifugation I get this viscous bubbly layer
floating on the top of the centrifuged lysate. There is a pellet therefore
it has been centrifuged properly. 

Has anyone had this problem and do they know what the layer is? I don't
think that it is unbroken DNA/histone as it has been thoroughly sonicated.
The layer is quite viscous which suggests its protein but it is in 9M urea
so it should have dissolved.

If anyone can suggest what this is then can you email me at:

g.s.stewart at bham.ac.uk

Thanks.

G.Stewart.



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