The simplest test of whether your CTD is dimeric is dynamic light scattering. This requires a special instrument. If all goes well, it will tell you the mass of the CTD protein in solution (only one of the CTDs is needed). Two other technique you could use to measure the mass of the protein in solution, if the equipment is available, are SEC-MALS (size exclusion chromatography with multiangle light scattering) and analytical ultracentrifugation.

As for your original question: The noncovalent dimer will almost certainly dissociate into the individual monomers on SDS-PAGE. You could use one of the tags to pull down the dimer, run the protein thus obtained on a SDS-PAGE, then perform a Western blot using an antibody that recognizes the other tag to show that the dimerization occurred. This is the same experiment suggested by Maxuel Andrade , but without having to make new constructs.

To add further confirmation, you could use a chemical crosslinker such as BS3 to make the dimer covalent, then do the pulldown again with the first tag and do the Western blot with the antibody to the second tag to show the doubling of the mass. The tricky part of this experiment is choosing the crosslinker concentration. The result is only meaningful if crosslinking occurs at a low crosslinker concentration, and preferably at a low protein concentration as well.

Another experiment would be to attach the protein by one of the tags to an SPR chip, then flow the protein with the other tag over the chip. This would not only demonstrate the dimerization, but would also give you a measurement of the affinity.

But there is a potential technical issue with experiments that rely on mixing the two CTD preparations together: If the protein dimerizes with very high affinity (Kd~pM), each of the two purified proteins will already be dimeric. Although the dimers would be in equilibrium with the monomers in principle, it might take a long time for dissociation of the dimers to occur. You would then have to incubate the two proteins for a long time to allow exchange of the monomers between the two dimers with different tags.

Hi,

you can make two constructions of your CTD: one fused to another protrein as GST or MBP that will be purified by affinity (glutathione or amilose resin, respectively) and other construction of your CTD with only 6His tag. After pull-down in glutathione or amilose resine your 6His-CTD should be observed on SDS PAGE or by WB with anti-6His antibody. Please, find a exemple of pull-down experiment with GST Tag in the manuscript 10.1111/mpp.12884 . Best wishes!

The simplest test of whether your CTD is dimeric is dynamic light scattering. This requires a special instrument. If all goes well, it will tell you the mass of the CTD protein in solution (only one of the CTDs is needed). Two other technique you could use to measure the mass of the protein in solution, if the equipment is available, are SEC-MALS (size exclusion chromatography with multiangle light scattering) and analytical ultracentrifugation.

As for your original question: The noncovalent dimer will almost certainly dissociate into the individual monomers on SDS-PAGE. You could use one of the tags to pull down the dimer, run the protein thus obtained on a SDS-PAGE, then perform a Western blot using an antibody that recognizes the other tag to show that the dimerization occurred. This is the same experiment suggested by Maxuel Andrade , but without having to make new constructs.

To add further confirmation, you could use a chemical crosslinker such as BS3 to make the dimer covalent, then do the pulldown again with the first tag and do the Western blot with the antibody to the second tag to show the doubling of the mass. The tricky part of this experiment is choosing the crosslinker concentration. The result is only meaningful if crosslinking occurs at a low crosslinker concentration, and preferably at a low protein concentration as well.

Another experiment would be to attach the protein by one of the tags to an SPR chip, then flow the protein with the other tag over the chip. This would not only demonstrate the dimerization, but would also give you a measurement of the affinity.

But there is a potential technical issue with experiments that rely on mixing the two CTD preparations together: If the protein dimerizes with very high affinity (Kd~pM), each of the two purified proteins will already be dimeric. Although the dimers would be in equilibrium with the monomers in principle, it might take a long time for dissociation of the dimers to occur. You would then have to incubate the two proteins for a long time to allow exchange of the monomers between the two dimers with different tags.

You can try crosslinking the protein by incubating it in presence of a crosslinker like glutaraldehyde (GCHO). Then run the crosslinked product on SDS-PAGE along with a marker and a negative control (protein without GCHO).