I have this recurrent problem with cytosolic protein extraction. The initial extract has all the proteins nicely solubilized, but upon storage at -20 over a period of time, a pellet forms, that doesn't dissolve upon thawing/vortexing. I have had some protein bands completely disappear due to this, and I think it's because some proteins are precipitating.
Has someone faced a similar problem? If so, could you please help me out?
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sedimentation of cytosolic proteins
Started by chembelle, Aug 02 2012 11:20 AM
cytosolic proteins Western blots sedimentation
2 replies to this topic
#2
Curtis
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Posted 02 August 2012 - 08:03 PM
Depends on the method of extraction and the buffers you use to keep them in. I use a digitonin extraction method and avoid using Dounce homogenizer. And I always keep my protein samples at -80C, not at -20C. And may I know what protein you are looking at in the cytosolic fraction?
#3
mdfenko
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Posted 03 August 2012 - 06:47 AM
some proteins can't withstand freezing. for this reason we add glycerol (to 50%) to extracts to prevent freezing at -20C.
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genius does what it must
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genius does what it must
i do what i get paid to do
Also tagged with one or more of these keywords: cytosolic proteins, Western blots, sedimentation
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