Can you speculate to the nature of the interaction? Is it transiently interacting or binding? If it is binding, could you have one protein immobilized on resin/beads via a tag (MBP, 6x-His, GST, etc) and perform an in vitro pull-down to see if the untagged, speculatively-interacting protein binds?

Thanks for you suggestion! We believe this is a transient interaction with relatively low affinity. We have already determined that these two proteins were interacting via NMR chemical shift perturbation. We just need another technique to validate the interaction and the native electrophoresis seams to be a good option. We have considered pull a pull down, but fear we might not be able to detect the interaction as it doesn't seem to be very strong at this point.

Got it! Transient interactions are tricky to nail down, good luck!

I also had trouble trying to run proteins with different pIs on native gels and unfortunately I don't have a solution. However, I was wondering if you could use gel filtration to show the interaction. The idea is to run the samples as individual proteins and mixed proteins on an fplc and observe a shift to higher molecular weights for the mix.

You can run native gradient PAGE electrophoresis. You can use gradient form 4-25 % PAA. Include the 12 kDa, and the 16 kDa proteíns, separately, and together (previously incubated together). Include molecular weight markers.

You will look for a new band of 28kDa, un the line where you include the two proteins being previously incubated together. Also, can appear bands with molecular weight bigger than 28 kDa,  in there are multiple interaction between the 12 and 8 kDa proteíns.

Yours,

Héctor Nolasco.

Consider chemical crosslinking followed by SDS-PAGE. Use a low crosslinker concentration, such as 10 µM BS3, to minimize non-specific crosslinking.

Hi Alex,

I agree with the previous suggstions. Pull-down assay and gel filtration are fast, easy to perform and allow to test different buffers, which may help in stabilyzing the interaction. It's possible, for example, that the pH8.8 of the buffer used for the native PAGE is not ideal for your proteins to interact. I would suggest you to try with pull-down first, as it requires low amounts of protein and you can test many different conditions and different ratios of the two proteins at the same time. If you don't get any positive result, I would go for crosslinking.

Regarding the native gel, I think it's actually difficult to predict a possible shift. The ability of the complex to run through the gel will depend on its net charge, which means that the "position" of the band of the complex may be different from both the single proteins.

Alex -

To study such interactions (assuming it is not too weak), you can try a sedimentation velocity experiment if you have access to an Analytical Ultracentrifuge (AUC). It is a "slow" method but provides a reliable orthogonal verification of non-covalently interacting species.

Hi Alex,

I have same problem ( Interaction between 2 proteins having different PI). Have you tried any new things please let me know.

Thank you

Hi Alex.  There are some good suggestions above and you pointed out one source of trouble yourself.  If you work at a pH below the pI of both proteins, they will both be positively charged.  If you work at a pH above the pI of both proteins they will both be negatively charged.  At the pI of a protein, it has no charge, so very close to pH 8, at pH 8.8, your protein of fixed concentration has a low but negative charge, and a decreased solubility over what it would have at pH's that are more distant from the pI. So, you won't expect to see a large gel shift if the charge is greatly decreased.  Binding to the other protein should drop the pI to about 6.8, approximately, because the two proteins will neutralize each others charge, but only for the time that the complex stays intact. 

I think that you would be better off working at one of three options: a pH above both pI's, a pH below both pI's, or a pH right in between the two pI's, i.e. at the average pI of 6.75, so both proteins would be nicely soluble and nicely charged, but one would be net positive and one would be net negative.

In fact, you could probably set the intermediate pH so that the 1:1 complex is exactly neutral, or very close to neutral, but 6.75 would be a reasonable first approximation.  You could also calculate a weighted average of pI's using MW to weight the individual pI's.  Then, if the 1:1 complex forms, it would likely precipitate and/or at least migrate very slowly on the gel, allowing you to isolate it by cutting out the band.

At a pH below or above the two pI's, this same 1:1 complex would migrate as a cationic 28 kD or anionic kD species, respectively, or a higher MW multimer, although to get this kind of migration, all you really need is a pH that is above or below the pI of the complex.  You do need to have the two separate monomers soluble enough to form the complex, however. 

I could imagine doing this reaction in solution and high or low pH and changing the pH to a more moderate value after incubating the proteins together, and then loading everything on a gel at the moderate pH.

I think that using precipitation, with luck, you could trap species that are only formed transiently.  They may even crystallize.  You may or may not be able to trap them with size exclusion, though running the column at low temperature after incubating the proteins together could slow dissociation kinetics down.

Best of luck.

Hi Alex！I've been looking for an experimental method for the interaction between two purified proteins by non-denaturing gel electrophoresis, but I feel that there are fewer literature on the native page of purified proteins, so can I refer to your protocol? Thank you so much!