By using SDS-PAGE we can find the proteins of various mol weight or size,
but can we find whether our protein binds to any other molecule and how efficiently it binds?
Also does anyone know any method to quantify insulin in our sample other than RIA?
I've found the easiest way to determine IF a protein binds another protein is by Co-IP. For a more quantitative method, I would suggest using the ELISA approach which is similar to immunoprecipitation but you can do a titration with different concentrations so as to determine how efficient they can bind. I've attached a protocol with some basic IP principals. Good luck!
If you're looking for a protein binding to a small molecule ligand, you might want to try fluorescence anisotropy. This technique measures how quickly a molecule spins around in solution. A small molecule will spin very quickly, and that spinning rate will slow dramatically when the small molecule binds to a big protein. If you label your small molecule ligand with a fluorophore, you can measure in real time how the ligand binds to the protein by monitoring the change in spinning rate (anisotropy).
for quantitation of the bond lifetimes and forces between a ligand-receptor pair, single molecule force spectroscopy techniques are used. For example, one covalently immobilizes the ligands on a surface and covalently immobilizes receptors on the tip of atomic force microscope probe, and then does a so-called "pulling experiment". Alternatively one can use an optical tweezer to study the interaction. If you do not need to know the forces of the bond but only whether the two molecules bind, you may be able to label each molecule with a donor or receptor and measure the FRET distance. These are all specialized techniques, so if you want something that is maybe easier to try in your own lab, tell us a little bit more about the molecules and what you have available.
If there's a tryptophan in the binding site it would also be possible to determine binding affinities through quenching of intrinsic tryptophan fluorescence. no labeling necessary
instead of RIA you could use enzymatic detection, for example ELISA
You can do SPR, surface plasmon rasonance to find the binding constant of any molecules with your protein of interest.
Thanks to all for your valuable answers.. And can u tell me more about how to quench intrinsic tryptophan?
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