protein precipitate while concentrating

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    • #7775
      ramen
      Participant

      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

    • #73624
      blcr11
      Participant

      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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