Hello all, so I'm trying to purify a protein complex with multiple subunits for the very first time, and after eluted with different concentration of imidazole there are multiple bands on my SDS-page gel. Mass spec result suggest that the desire subunits are all present, but it's hard to identify which is which on a gel and they may not run at the expected size. I was wondering if there is a way to identified each subunit? The last subunit is tagged with His tag, however the western blot on that one is not really successful as the expression is very low. We also tried GFP tag, but the signal seems to be coming from free GFP only. I am not sure what else we can do to identify the subunits, or pull down the whole complex before purifying?
You can excise individual bands from the SDS-PAGE and send them to mass spec for peptide sequencing, this way you can identify each of the bands individually without having to do extensive cloning and pulldown assays
It may be silly, but have you tried computing the "expected size" of your different subunits ? (https://web.expasy.org/protparam/)
By running your elution fractions next to a protein size standart, you should have a pretty good idea of which band corresponds to which subunit, given that they have different sizes.
If you have too many bands, you can always try a higher concentration of imidazole (say 500mM). Only the his-tagged proteins should remain bound, so look for the sharpest band(s) left.
I would verify that the bands are not due to non-specific binding first. Gradually increase the imidazole concentration in the wash buffer and see if the band pattern changes. You can also try running a native gel to see if the proteins separate or remain in a complex. Separation would suggest non-specific binding.
If you have purified protein for any of your expected complex bands, run them alongside your sample to identify the bands in your sample.
Does your complex do any chemistry? If so, use your sample in a reaction and see what products you get.
There are other florescent tags you can use. Look at this paper for details:
Wombacher, Richard, and Virginia W. Cornish. "Chemical tags: applications in live cell fluorescence imaging." Journal of biophotonics 4.6 (2011): 391-402.
Martin Chenal Hi Martin, thank you very much for your reply! Unfortunately the complex we are working on is membrane bound and from what I've been told membrane protein may not be running at predicted size/ or they might run in dimer etc. But thank you so much for the useful resource!
- Badges
- Science method