Dear all,
I am trying to check the interaction between two of my proteins of interest. However before trying any biophysical technique such as SPR/ITC/MST etc, I used normal superdex-200 gel filtration chromatography. I used protein A (Receptor; ~40 kDa) and protein B (Ligand; ~31kDa) in purified form and mixed together with 1:2 molar ratio at temp. 25 degree for 1 hour. Now when I performed Superdex-200 with three individual runs (Ligand, Receptor and Complex). each time I get a surprising result wherein the complex has more retention volume than the Receptor it self (attached result brown: complex, cyan: receptor and blue: ligand).
When I run the peak fractions from each run through SDS PAGE, I get the two different protein bands in the complex run (attached result; lane 1-3 is the Ligand and Lane 4-6 Receptor and lane7-9 Complex peak fractions). Please suggest what could be the possibility of such result. The complex formation shows about 1 degree (57 to 58 degree C) rise in melting temperature every time as compare to their individual ones. Buffer used for the protein are same 1X TBST pH=7.6.
There is not much difference in elution time between the three samples. The small differences may not be significant. Although your expectation is that the complex would elute earlier than the individual proteins, this might not happen for a variety of reasons: (1) No stable complex was formed, (2) the complex formed, but it eluted at about the same time as the individual components because of a more compact shape of the complex compared to the individual components, (3) at least one of the component proteins interacts with the column resin strongly enough that this interaction dominates the elution profile of the complex so that it elutes at the same time as that component.
Hi there,
Did you calibrate the column? If yes what is the apparent MW of each peak? One possibility is that receptor and ligand alone are both dimer and complex is a hétérodimer.
Dhiraj Srivastava
Yeah True, I am also worried about it that being 40 kDa, it came early in the elution volume that reflect the formation some kind of oligomer. What Adam B Shapiro is suggesting seems very very logistic to me. Those reasons may be the possible cause of such behaviour.- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology