Hello, I am looking to do in vitro crosslinking using formaldehyde and DSS to determine if two recombinant proteins of interest form a heterodimer. We also anticipate homodimers. I originally planned to do single protein controls for each proteins to compare to, but I would like to take into consideration non-specific crosslinking that might just be due to concentrations of protein/crosslinker. We plan to analyze the samples using NativePAGE western blotting and silver staining. What controls are necessary to definitively conclude that our two proteins are binding to eachother? Many related studies simply show the heterodimer vs uncrosslinked or single protein controls.
With regard to the chemical crosslinking study, an approach you can take is to minimize the protein concentration as far as possible during the crosslinking. You can also test a range of crosslinker concentrations. If the two proteins molecules are truly bound together you should see crosslinking persist even at very low protein and crosslinker concentrations. At higher concentrations, you should also see non-specific crosslinking.
Beyond chemical crosslinking experiments, you can show that the proteins elute together from a gel filtration column. You can also do an immunoaffinity pull-down experiment, whereby you use an antibody against one of the proteins and show that it pulls down both.
Design of the experiment will depend on exactly what you're expecting. You say that you anticipate homodimers as well. Is the final complex expected to be a dimer of dimers, or is there a competition between homo- and heterodimerization?