I need to separate small protein complexes in the range of 50-60 kDa with 10-15 kDa difference. Has anyone worked with something like that? Are there gel filtration columns available in that size range with adequate separation? Would a native gel work and with which percentage?
Thank you for suggestions!
If you have a Fast Protein Liquid Chromatography system, you can separate these proteins using a size-exclusion column. There are columns that separate proteins in this range, for example, GE Healthcare Sephacryl S-100 (range: 100000 Da - 1000 Da). However, if your proteins do not denaturate in organic solvents, you can separate them using a reversed phase chromatography, using, for example, a C18 column. In my opinion, this is the best way to separate these proteins.
Ya there are Size exclusion columns (superdex, sepadex) available to separate protein complexes in a wide range. You just got to choose the right one for your need. http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-de/applications/gel-filtration-gf#
I am not separating proteins, rather protein complexes, so SDS PAGE is obviously not a solution. I did check data sheets for several size exclusion columns, but I would appreciate personal experience on them.
You can use a tricine gel at 12% (or lower). You will be able to separe proteins with a difference of 5 KDa
I think I cannot stress it enough: I am NOT separating proteins. I am separating complexes. Complexes that fall apart in SDS, as most of the complexes should. And therefore neither SDS PAGE nor Tricine SDS PAGE can be an option
We have already told you: size exclusion columns, using hplc or fplc system. But which column you're gonna use is up to you. It depends on which kind of complexes you have. Therefore, now you have to find the appropriate column for your case.
Hi Maria, are you looking to separate these protein complexes in order to study them in a gel or in solution?
Native PAGE gels can be used to study protein interactions in the absence of SDS, but since native PAGE separates proteins based on size and charge, you will have to test different gel preparations that give you the best separation. I agree with Taverniti, in that 12% native PAGE gels might be your best bet.
Gel filtration (size exclusion) will not completely separate proteins (or complexes) that are that close in MW.
Anyway, your choices for separating these protein complexes depends on how stable they are. If they are stable, (Kd< 10e6) then you can explore ion exchange chromatography. If the complexes are unstable (Kd> 10e6), then you are limited in your choices.
As said before, size exclusion columns IS the solution because you can peform them in NATIVE buffer, and there is no SDS in it (it's not a SDS Page). So your complex may stay intact if you choose the good buffer. But you will need a Fast Protein Liquid Chromatography (FPLC) system like an "Akta" and the good cutoff range size exclusion column (or ion exchange, or hydropathic or any other suitable column ... protein purification needs often several trials).
If you want to perform only the simplest purifications or if you have no acces to FPLC, you can also tag one of the protein of your complex with an His-tag (be carefull of where you put your tag to maintain the complex ok) and and use gravity His-trap column : once again that's purifications in (almost) Native buffers