Hi everybody,
There is one aspect that troubles me with regard to protein-ligand separation.
Gel filtration separates molecules by their stokes radius, and is a powerful technique for buffer exchange (so is dialysis). But gel filtration is used to determine oligomeric forms of proteins too !
I have read that gel filtration was used to separate proteins from small ligands, but I wanted to know at which dissociation constant we can have a full separation between the two (or between two proteins).
I think the answer is not very straightforward, but maybe you know a publication about this problem that I didn't find ?
Thank you !
There is not a set Kd marking a boundary between full and partial separation. Rather, the higher the dissociation constant, the better the separation, regardless of method (dialysis or GF). You can use conditions that lower the affinity of the interaction (pH extremes, chaotropes, high or low salt) and then perform the separation, if removing all ligand is really important.
In your case the problem may not be dissociation constant, but the half life of your complex. If that is long compared to the time needed for separation, you can separate protein-ligand complex from free ligand.
One common method is spin columns: 1 ml Sephadex in a tuberculin syringe. Add the sample on top and spin through the column in a table top centrifuge. Protein will come with the flow through, free ligand will be retained on the column.
Another trick is to suck the sample through a PVDF membrane on a vacuum manifold. The protein will be retained on the membrane, the ligand passes through. With luck, your complex is stable enough for a quick washing to keep the background low. I have published that method with both Na/K-ATPase and Hsc70.
Based on the work on chloroplast F1 ATPase nucleotide binding by Bruist and Hammes (1981), ligands bound with an equilibrium dissociation constant in the single-digit micromolar range will dissociate readily during gel filtration. More tightly bound ligands may not be completely removed, although I imagine it would depend on the length of the column and the flow rate.
Thank you for all your answers, my dissociation constant seems to be around 50 µM, so your comment is encouraging Mr Shapiro.
Mr Buxbaum, effectively the koff must be the principal parameter for a good separation, unfortunately I don't have this information.
About the PVDF membrane method, isn't it destructive? My goal is to keep the protein in good shape without the ligand.
Thank you again !
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