protein precipitation upon dialysis - (Nov/29/2006 )

Hi,

I have used 8M urea extraction buffer(with 20mM Tris and 0.5M NaCl,pH7.9) to make a protein homogenate.However, the proteins got precipitated when i dialysed against it Tris buffer made at the same concentration as used in the initial urea extraction buffer.i would like to know if i can go ahead with doing western blot with the precipitated sample I have?I have a large voulme(30 ml) of the homogenate now.Should I concentrate the homogenate by speedvac and then dialyse against low molar urea concentrations or should i do dialysis and then concentrate the sample?

Thanks,
Nirupa S Mogili

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you have to check which proteins of your homogenate are precipitating during dialysis beside your are working with a homogenous protein solution

to me it is unclear how the dialysis buffer is composed and conditions of dialysis are arranged (temperature? stirring? what should be lost during dialysis?)

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Try dialyzing against a different buffer or lower molar concentration buffer before doing anything with the antibody. This has happened to me before. Try 50mM bicarbonate buffer.

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the dialysis was carried out at 4C with magentic stirring.i used 20mM Tris at pH 7.9 as dialysis buffer

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so, it should be the urea that dissolves your proteins; proteins precipitate when losing urea; you can try to dialyse against 20 mM Tris and 2 M urea and may succeed without precipitation but you may not circumvent to have some urea in your buffer to meet solution of your proteins...

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well at 8M, urea precipitates itself at 4°
if you want to do at 4°, i know (98% sure) that 4M is the maximum

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