Protein pellet solubilisation - (May/02/2003 )

I have been extracting proteins from rat brain, heart and kidney for use as positive controls on my Western Blots. I have precipitated the protein from the supernatant using TCA and the resulting pellets are washed with ether.
The problem is that some of the pellets will not solubilise into 1:1 sodium carbonate:DTT even under sonication. Does anyone have any ideas as to how I can get these proteins into solution?

Thanks

Rachel

-floyd78-

I had this same problem last year while resolubilising protein pellets from the fundus of the stomach. I tried DTT, urea and finally it solubilised in 6M guanidine HCL. however, i don't recommend you try this as my western didn't work at all due to thepresence of too many salts. Supposedly this was due to lots of mucous proteins produced in the stomach which protect against acid, anbd i should have tried some other step during extraction. can't remember what it was exactly but something to do with removing those proteins in hte early stages which could interfere. i htink once you are in this stage there is not much you can do but get more tissue and start again. good luck!

-hellsy_x-

QUOTE (floyd78 @ May 2 2003, 11:30 PM)

why not trying Laemmli sample buffer directly?

if too much is still insoluble, add a very little amount of DMSO before using Laemmli-buffer;

heart cells are more critical, I would not heat but incubate 1h at 37°C (add proteases inhibitors)

-The Bearer-

QUOTE (hellsy_x @ May 28 2007, 02:45 PM)

Your western did'nt work because of guanidine precipitate in SDS containing Lemmly buffer, you should dialyse your protein before SD PAAG , solubilized in GuaHCl against 6-8 M Urea , in many cases it works good and no precipitation occur

-circlepoint-

Hi Rachel, you could try urea 7M/thioure 2M/detergent 1-2%. This is what we use for alot of 2D PAGE work - many things, for example plasma proteins, won't dissolve in anything else once precipitated by TCA.

Andrew

p.s. the detergents we use are CHAPS, C7, ASB-14, etc...you would have to check the solubility of others in that brew.

QUOTE (floyd78 @ May 3 2003, 07:30 AM)

-Andrew1-