Hi there,

Gel filtration shows your sample oligomerization state. If your protein was monomeric in solution you would get a unique peak corresponding to the MW of the band seen in SDS PAGE. From your experiment you can conclude that your sample is not homogenous. Part of the protein is monomeric (peak3), part is oligomeric (peak 2, dimer?) and part is aggregated if peak1 corresponds to dead volume. I don't think there is a peak4 but just the rest of peak3. If interested in the monomeric form, you should purify it from this experiment by collecting fractions of interest.

Whats the SEC matrix? This defines the resolving power of the column and will help with interpreting the chromatogram. First guess is that you have a fair amount near the void volume in peak 1 but you have to know the void volume of the column to be sure . Its usually about 30% of the column volume (Cv), but a 2MDa blue dextran run will better define this. Its possible you have monomer/dimer and trimer represented in peaks 2/3/4 but a bit more data is needed. Gel is denaturing and reducing I assume?  Protein looks nice and clean but its native solution behavior can be quite complex.

With  the help of SDS-PAGE & gel filtration you can be assured that this is the molecular weight of sample you loaded. Interestingly in filtration you got three peaks that shows that your protein  is polymeric  that has combined MW of 66kd. YOU can collect the each peak separately  and run in  PAGE  in different lanes  that will tell you than exact weight of different fragments. For much information you can run 2D gel. 

I missunderstood my question. I obtained gel filtration before proceeding SDS-PAGE. So my question is " After obtaining SDS-PAGE result, how can we know that the clear band with MW around 66 kDa come from around fraction 47?"

Hi there,

Strange question... The SDS/PAGE pattern comes from the material loaded onto the gel. Obviously the only protein material in your sample is something running like a 66kDa protein when denaturated. To have a clearer picture of the situation, you should run every peak obtained on SDS/PAGE to compare their respective contents. The other thing to do is to calculate from column calibration what is the apparent native MW of the material present in fraction 47 (Is it 66kDa or else?) and in other peaks.

If available, an independent assay system such as immunoprecipitation or biological activity would help identify the fractions containing the protein of interest.

It is difficult to correlate other than by running the peak samples on a gel because gel filtration assumes a globular conformation and the same protein size in a rod shaped conformaion will appear to be a different molecular weight as Dominique suggests