I have separated soluble and insoluble fractions of two proteins that differ by some amino acids. On the picture 1 - total, 2 - insoluble and 3 - soluble fractions of one protein. 4, 5 and 6 refer to other protein. In the first case the insoluble protein has a much lower concentration that in the other.
One thing that might cause this is that I washed the precipitated insoluble protein before dissolving in urea. May washing cause solubilizing of insoluble protein?
You state that: "two proteins that differ by some amino acids". Even a single amino acid change can affect protein folding and solubility so unless you re-do the experiment with the same protein you can't really draw any conclusions.
Insoluble protein is not always in inclusion bodies and may sometimes be resolubilized by dilution in a suitable buffer. Salt concentration can be an important factor.
I agree with the before written. In addition: you will always have a loss (of protein) after a washing step, even if the protein is not solubilized by the washing buffer. If solubilization by the washing buffer is possible at all is another question. Depends on your buffer, but with "normally" use buffers I would consider this as very unlikely. Depends also, if you have your insoluble protein partly not located in inclusion bodies and maybe not insoluble but more or less "poorly" soluble.
(most proteins are soluble, although not in aqueous solutions :-) )
Unfortunately I don't have time to repeat all from the beginning. But I tried to run another gel with the same probes, hoping that in one of them precipitate wasn't dissolved enough.. And it helped! God exists, now I can print my thesis out)
Thank you for answers
Esther Breslow Weill Medical College of Cornell University
I agree with most of the previous comments., particularly the first two. Let me add that, in my experience, insoluble proteins can most often be solubilized by changes in solvent pH. Changes in temperature might work in special circumstances, but runs the risk of causing irreversible denaturation.
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