fractionation of complex protein mixture for ligandscreening

[mbsmeets]

Could anyone point me into the right direction for fractionation of our complex protein mixture that we want to screen for the presence of ligands and antagonists for a specific receptor. We need to reduce complexicity of our sample and were thinking of using anion exchange to get for example 20-40 fractions (probably still mixtures of many different proteins, but that is okay). These fractions will then be tested for the presence of a ligand or antagonist using cells overexpressing the receptor. Positive fractions will have to be further screened of purified to identify the ligand but that is of later concern.
Are there any (mini spin) columns that can be easily used or do you need advanced HPLC- or FPLC-systems

[unmgvar]

what are the concentrations you are working with?

can you right now seperate your samples somehow?

you could use a 2D solution with HPLC for the separation and then collect the fractions if they can be seen in UV.

i am guessing that under the correct conditions the chromatography will not effect your proteins too much. but i have not enough experience here on that matter. maybe someone else can comment on the feasibility of my proposal

[Mark Tracy]

You will need HPLC. Spin columns don't have enough resolution to get 20 fractions. (Personally, I find the risk of blunders with spin columns excessive. Maybe I'm sloppy.)

Since biological activity is essential, you should use only methods that preserve activity. (Reversed-phase is out of consideration.) Ion exchange (WAX, SAX, WCX, SCX) or hydrophobic interaction chromatography are the way to go. (May I recommend Dionex ProPac and ProSwift columns? See www.dionex.com)

Ligands and receptors commonly have post-translational modifications, especially phosphorylation. This makes them sensitive to metal contamination in the HPLC system. Use a bio-compatible system.

Don't worry about detection. Just collect fractions. Your cell-based assay is the detector. Later, you can go back and try to isolate the interesting proteins, and you will need LC/MS at that stage.

Best of luck, you have an ambitious project ahead of you.