cell lysate problem

Matthew Parker MatthewP at microbio.uab.edu
Sun Jul 19 15:13:14 EST 1998



Grant Stewart wrote:

> 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.
>
>


    Is it lipid?




















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