Hey everyone,
I'm working with a protein that I managed to purify to ~95% purity as estimated by SDS PAGE. The protein seems to be stable for at least a week however after that time it loses activity. Out of interest I ran an SDS PAGE again on the sample and noticed it had broken down into distinct domains This is in the absence of a protease (I think). I'm also seeing some evidence my protein could be a part of a complex. I have one or two things I wanted to ask:
Thanks again for your help everyone
Harry
Hi there,
There are definitely some protein structures which exhibit an increased resistance to the usual denaturation process (SDS+temperature). Addition of 8M urea to sample buffer may help to solve the problem.
There is always a linker between protein domains. Due to its flexibility and exposure to solvent, the linker is more susceptible to degradation (spontaneous or protease mediated) than the rest of the protein. About protease, you can't be 100% sure the degradation you observe is not due to protease contamination as it works at catalytic dose which is difficult to detect.
By boiling your sample in SDS containing buffer, all non-covalent interactions would be broken apart.
If you purified your protein in a complex, and the interactions between your protein and its partners are non-covalent, you would have multiple bands to begin with.
If I am not using a protein immediately, I always snap freeze it and store it at -80C. Or store it in -20 with some glycerol (and test if it retains activity).
Single band on SDS-PAGE can be deceiving. If you load your protein in a higher concentration, you will probably see many more bands showing up. I once crystallised a "single band" protein which turned out to be a very faint band in the background.
There might also be a chance that microbes broke down your protein if you leave it at room temperature/fridge over a week.
Do you run your gels reduced versus non-reduced? When you run gels non- reduced do you include an alkylating agent? There could initially be some internal clips held together by disulfide bridges but the clipping gets worse over time. Apparently you initially had 95% of one band and ~5% of another band(s) and then ran the gel assay again when the activity was lower and found multiple lower bands, right? If you did a silver stain you will see even more bands and with overloading you might see a trace amount of a protease. Keep in mind that the pKa of your SDSPAGE sample buffer changes dramatically with temperature and therefore the pH changes where certain peptide bonds are labile such as DP sequences. What is your formulation buffer and storage condition? If your preparation was that pure then your putative degraded sample would lend itself to LC/MS to see if the supposed subunits are actually fragments of one polypeptide chain. Do you have access to a triple quad MS or can you blot to a membrane and cut out the bands for gas phase amino acid sequencing for N-termini?
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