What methods can be used to study a competitive interactions between two different proteins (eg. A & B) for a common binding motif on protein (C).
1. Fluorescence resonance energy transfer (FRET). Attach a fluorescent donor label on protein A and acceptor label on the peptide (or vice versa) such that there is FRET between the labels, as measured by fluorescence intensity, when the peptide is bound to A. If B competes with A for peptide, then the FRET signal will decrease as the B concentration increases.
2. Chemical crosslinking. Use a chemical crosslinker that causes covalent bond formation between A and peptide. This could be an exogenous compound, or the peptide could be synthesized with a reactive group. The reaction could be observed by a change in mass of A on SDS-PAGE, if the peptide is large enough. Determine whether adding B results in less crosslinking of A with peptide accompanied by an increase in the mass of B. Intact protein mass spectrometry would be a more exact method of demonstrating this. In either case, it relies on there being a substantial mass difference between A and B.
3. Surface plasmon resonance (SPR). If there is a substantial mass difference between A and B, then SPR could be used to distinguish between A and B binding to immobilized peptide by a change in the size of the resonance signal.
It might not be necessary to do this experiment as a competition between A and B. It might be enough just to measure the Kds for the peptide-A and peptide-B interactions separately. This could be done by a number of different methods, including isothermal titration calorimetry (ITC), differential scanning fluorescence, FRET, fluorescence polarization, equilibrium dialysis, SPR, microscale thermophoresis, intrinsic protein fluorescence, etc.
1. Fluorescence resonance energy transfer (FRET). Attach a fluorescent donor label on protein A and acceptor label on the peptide (or vice versa) such that there is FRET between the labels, as measured by fluorescence intensity, when the peptide is bound to A. If B competes with A for peptide, then the FRET signal will decrease as the B concentration increases.
2. Chemical crosslinking. Use a chemical crosslinker that causes covalent bond formation between A and peptide. This could be an exogenous compound, or the peptide could be synthesized with a reactive group. The reaction could be observed by a change in mass of A on SDS-PAGE, if the peptide is large enough. Determine whether adding B results in less crosslinking of A with peptide accompanied by an increase in the mass of B. Intact protein mass spectrometry would be a more exact method of demonstrating this. In either case, it relies on there being a substantial mass difference between A and B.
3. Surface plasmon resonance (SPR). If there is a substantial mass difference between A and B, then SPR could be used to distinguish between A and B binding to immobilized peptide by a change in the size of the resonance signal.
It might not be necessary to do this experiment as a competition between A and B. It might be enough just to measure the Kds for the peptide-A and peptide-B interactions separately. This could be done by a number of different methods, including isothermal titration calorimetry (ITC), differential scanning fluorescence, FRET, fluorescence polarization, equilibrium dialysis, SPR, microscale thermophoresis, intrinsic protein fluorescence, etc.
The above answers by Adam B Shapiro are already good ones.
- You could carry out a simple invitro pull down assay to get an idea if the two proteins (A&B) are competing for same binding site on protein C. Tag one of the proteins (C) and increase the concentration of one of your binders (A or B) gradually in a same reaction mixture. Run the reactions on SDS PAGE and you would see the depletion of one of the binders. This would be a qualitative assay.
-In order to get affinity values you could try ITC displacement assay. Reference paper for this technique Article Single-experiment displacement assay for quantifying high-af...
This technique would be an useful one if the displacement of one of the proteins by the second one takes place in the same molecular pathway.
Thank you everyone. I am about to start the study and will be trying each method suggested by you in my experiment.
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