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Redissolve dry protein pellet in RIPA buffer

western blot protein RIPA buffer pellet

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#1 elmariachi

elmariachi

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Posted 02 November 2011 - 01:47 PM

I'm trying to extract proteins from white adipose tissue to run a Western Blot.
I'm using a modified Bligh and Dyer method. At a glance: tissue is homogenized in a solution of RIPA+chloroform+methanol, centrifuged to obtain 3 phases (a lipid phase which is discarted, a protein "disk" which I'm using and an aqueous phase which I'm also using). Then the proteins from the "disk" are washed with acetone twice and the resulting dry pellet is redissolved in RIPA and stored for quantification. The proteins from the aqueous phase are precipitated using TCA overnight, centrifuged and washed like above.

My samples are stored in 500ul (i think..) eppendorfs and since they weren't well redissolved a pellet has formed in all of them. Now I have two questions:
1- Can these samples be used and how can I make them usable (i.e. dissolve the proteins properly).
2- What is the correct procedure to redissolve a dry protein pellet, for future reference.

Thank you very much!





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