Hello all,
I was wondering if I am in danger of crashing out my protein of interest (native fliC) under these conditions:
first, I solubilized E. coli cell lysate pellet with 8M urea + 200mM acetic acid. pH 4. I subjected the soluble fraction to 14% PEG3350 precipitation. Next step in the schematic is to perform 2-phase separation using TritonX-114 and PEG3350. But before I performed 2--phase, I dialyzed my protein sample (after PEG) in 20mM Tris, pH 8.0 (buffer that I will use to equilibrate my anion exchange resin-- after 2-phase).
Was this a smart thing to do? Or am I in danger of precipitating out my proteins with such abrupt buffer change? I have read in Methods in Enzymology by Dr. Burgess that denatured proteins at 8M Urea need to be dialyzed step-wise in decreasing molarity of urea to avoid misfolding..
Best,
an undergraduate researcher with many questions
Every protein behaves differently. Dialysis is a slow process but the refolding process has to occur in a shorter time otherwise you will see precipitation.
If your protein was precipitated after being in pH 4 buffer, there should be very little of this low pH buffer left in the precipitate. You can dialyze the protein in the pH 8 buffer, but I agree that you should not go from 8 M urea directly to a buffer without urea because your protein will have a higher chance of precipitating or misfolding. By stepwise lowering of the urea concentration, you allow the protein to fold slowly, which is important to promote correct folding. It is usually better to slow down the folding, especially in the absence of chaperones in order to allow the protein to fold correctly and not simply collapse in an improperly folded structure or aggregate. The slow dialysis and stepwise dilution of the urea actually helps promote correct folding. good luck and remember that there is still a chance that your protein may not be correctly folded even after doing these steps because the pH during extraction may have altered its structure. However, if this is an established protocol for the extraction of your protein, you will be fine. Protein activity and CD spectra are two techniques to help you verify, directly and indirectly, respectively, that your protein is folded correctly and active.
Anjello
I wonder why do you solubilize the cell pellet in 8M urea, and why pH 4. If it is in inclusion bodies, you are better off lysing and removing soluble fraction, dissolve in urea, and take it from there. You will get rid of 90% of the impurities.
As Omar says, every protein is, I should add, VERY different. Some behave beautifully after refolding by dialysis ( I assume your protein is UNFOLDED), but many require dilution. A third group, most of the proteins that go to IBs, require effort and sometimes luck.
If the protein is unfolded, your best option will be to do the ion exchange in urea, it will come out pristine if you get the gradient fine. Once pure, you devote your time to refold it.
Among the million different ways to do this (once you ruled out dialysis) is by dilution on different buffers. First, look at things like pI, number of cysteins. You can run 96 plate tests modifying parameters, looking for absence of turbidity (say at 400 nm) in any standard ELISA reader. That means you have at least soluble protein. Run a CD and check whether this is the case
The next alternative is to use a crystallization kit. The next one, depending on your belief, go to church and pray.
I forgot, you may want to consider fusions. Unless it has lots of cys, use MBP, it raises the dead. Ifit has lots of cys, go for GST
By the way, your 2-phase separation looks great, we should use it
Dear Anjello Luciano You can dialyze the protein in the pH 8 buffer, but I agree that you should not go from 8 M urea directly to a buffer without urea because your protein will have a higher chance of precipitating or misfolding. By stepwise lowering of the urea concentration, you allow the protein to fold slowly, which is important to promote correct folding. It is usually better to slow down the folding, especially in the absence of chaperones in order to allow the protein to fold correctly and not simply collapse in an improperly folded structure or aggregate. The slow dialysis and stepwise dilution of the urea actually helps promote correct folding. good luck and remember that there is still a chance that your protein may not be correctly folded even after doing these steps because the pH during extraction may have altered its structure. However, if this is an established protocol for the extraction of your protein, you will be fine.
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