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Did you filter your lysate through .45 then .22 filters? cheers b Quoting James Stroud <[log in to unmask]>: > Try refolding before purification. > > > On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: >>> Hi all >>> >>> sorry, for offtopic query... >>> >>> I am trying to purify my protein by Ni-NTA affinity chromatography. After >>> sonication as i centrifuge bacterial lysate, soon after 10 min >>> whole lysates >>> get precipitated during loading on the column and some time it remain >>> soluble too. if i get purified through the column without >>> precipitation, it >>> gets precipitated during dialysis. >>> I have tried lot, by chnaging buffers, increasing salt or deacreasing salt >>> or no salt at are helpless. >>> I do purifiaction in cold room. >>> >>> can any one suggest some solution? >>> >>> Thanks in advance. >>> >>> NSH >>> > > -- > James Stroud > UCLA-DOE Institute for Genomics and Proteomics > Box 951570 > Los Angeles, CA 90095 > > http://www.jamesstroud.com >