protein precipitate while concentrating

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ramen
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protein precipitate while concentrating

Post by ramen » Wed Jun 06, 2007 7:25 pm

protein in buffer 50mMTirs-Cl, pH8.5, 250mM NaCl was getting precipitate while concentrating. increasing salt to 500mM doesn't help. And I tried 10%glycerol in buffer, still doesn't work. that's what I can do to increase the solubility. anyone else has any other method to increase the solubility of protein? I do need high concentration of protein.
thanks

blcr11
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Post by blcr11 » Thu Jun 07, 2007 5:17 pm

More typically proteins like a little salt around, but that's not always the case. And what is the pI of the protein? If it's near 8.5 you might want to move away from that pH--maybe to pH 6.5-8.0 range somewhere. Could try going down in salt, like I said, to like maybe 50 mM and see if that helps. You could consider changing the buffer; Tris is the kind of buffer that will interact directly with proteins. You could try Bis-Tris, or HEPES, or even possibly imidazole--imidazole also can interact directly with proteins, but it's one of those funny kind of buffers (like cacodylate): more often than not it's either a negative or, at best, neutral factor, but when it works, it's like magic. Less likely addivites to try when you get sufficiently desperate are things like non-ionic detergents or 5% DMSO--or some mixture of any or all of the above. How sensitive is the protein to air oxidation? Might also consider adding DTT or something similar.

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