I am considering assaying oligomerization of my membrane protein. I am wondering if it is possible to run a clarified mammalian cell lysate over a gel filtration column using FPLC. We have the ability to reverse the direction of the flow and so assuming that the complex lysate mixture will likely dirty up the column, we will plan to reverse wash the column after every run.
The only hitch is that you will encounter is that you will be monitoring the elution of the proteins at a wavelength 280nm so you wont know which is your protein and therefore not know the oligomeric state. The way to do this experiment is to use a GFP-fusion protein (or equivalent). This technique is called fluoresence size exclusion chromatography (FSEC) and was developed by Kawate and Gouaux (see there 2006 paper in structure). There are dedicated columns for this and cleaning-in-place is essential. Hope that helps.
Hi Charles,
Gel filtration is a polishing column. Try perform first, a affinity column or anion exchanging, only after use gel filtration, because the column life time will be lesser, and your purification results will be poor.
Good luck.
Fábio.
Since you are working with a membrane protein, you may tray a detergent extraction for your protein an then run into the SEC. But, as Fábio sed, this is a polishing step so, a previus porification step is recomended if you are planing to use that column several times. Another thing: Be careful with hidrophobic proteins on SEC, sometimes they interact with the matrix. To avoid this you should add detergents (like S.D.S. or tween) into the buffers
with membrane proteins, there is almost never any resolution with gel filtration. Everything comes out in the void volume. This is because everything needs to be done in the presence of detergents. They are pretty much worthless for what you are trying to do.
If you decide to go that route, I would at least suggest a denatured injection onto a column in a native buffer system and possibly running the column buffer with 8M urea or 6M GudHCl and lowering the concentration with each injection to view the oligimer. Why not purify the protein first though under denatured conditions and then run a folding/oligimer assay on the column?
Article Use of fast protein size-exclusion liquid chromatography to ...
Try it, but there is a chance you'll have to throw away your column afterwards, or that it has lost some resolution. You may get local high pressures, overpacking the gel filtration material at the top of the column. Reversing the flow after the experiment may not resolve this. Detergents may not be so bad for the column, lipids may stick irreversibly.
It is actually common practice to run crude cell extracts on SEC columns (carefully not overloading the column..), but then using FSEC as Luke pointed out earlier. An option to having a GFP tag is using a fluorescent probe that binds to the his-tag of your protein. This route is very common in checking for which detergent to use in the following purification.
However, if you wish to do this to evaluate the oligomeric state of your protein it will be almost impossible to get any secure number out, unless you have a very large protein that is stable in a small size micelle forming detergent like beta-OG.. You will be looking at a protein-detergent micelle complex that will elute at a much larger apparent molecular weight than the protein itself.
To truly get a good result for the oligomeric state, you would need to purify the protein and then use eg SEC-MALLS to calculate your oligomeric state.
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