I agree with Valentina and Andrea, it would be best to have purified proteins and use biophysical approaches to obtain the binding constant (such as ITC, SPR or MST). If you use MicroScale Thermophoresis (MST), you could try to run the interaction study in cell lysate as well. For your setup, I would suggest to express RUNX1 as a fluorescent fusion protein, just lyse the cells and run the experiment with a dilution series of purified CBFb or CBFB-MYH11. This way you would obtain the binding constants in very close-to-native conditions!

Blotting is difficult to get to be quantitative.

If you have specific antibodies for each CBF isoform that you can standardize to the target concentration, you can try a competition blot like that from biological extracts by pulling down with an affinity tagged RUNX1 (or use NHS-activated sepharose to make your own RUNX1 resin) and blotting for both isoforms.

Alternatively you could try to do a more biophysical approach like fluorescence monitoring (if you have appropriate labels) or ITC with isolated proteins.

Dear John,

as Valentina stated, you cannot really assess affinity binding constants unless you have access to purified amounts of your proteins of interest, obtained from cellular extracts or, even better, from recombinant production. In this case, it could be highly recommended for example to run Surface Plasmon Resonance in order to measure “pure” affinity constants.

That said, you can surely try to extract some bits of qualitative information from your immunoprecipitation experiments. If you can do that, I would dare to suggest you to immunoprecipitate via anti-RUNX1 and then detect via Western blot for the two ligands. I believe that the fact that you would detect both the isoforms would imply at least that the two binding affinities are likely to fall in a similar order of magnitude range. If CBFB-MYH11 is really displaying more affinity than CBFb, than you may hope to detect more signal for MYH11, but be careful that the signals you get from Western blots can be really misleading from a quantitative viewpoint. The really important parameter is the amount of the two ligands that you have exactly in your sample. When you try to estimate binding constants you should always have an idea about the concentration of your ligand. In other words, one ligand can have a much larger affinity than another one but if it is present at a very low concentration you can struggle to see some binding at all.

As a paradigmatic case, you may think about hemoglobin and the partition between carbon monoxide and oxygen binding.

Good luck!

I agree with Valentina and Andrea, it would be best to have purified proteins and use biophysical approaches to obtain the binding constant (such as ITC, SPR or MST). If you use MicroScale Thermophoresis (MST), you could try to run the interaction study in cell lysate as well. For your setup, I would suggest to express RUNX1 as a fluorescent fusion protein, just lyse the cells and run the experiment with a dilution series of purified CBFb or CBFB-MYH11. This way you would obtain the binding constants in very close-to-native conditions!