Yes, that's quite possible. I'm assuming you are running WB with anti-His AB, right? First, I would run WB with the unfolded flow-thought fraction of sample B from the affinity column. Second, try to unfold (destroy the complexes or even denature a bit your protein) the sample B BEFORE loading to Ni-NTA (or whatever you have for His-tag) column. And the last suggestion, I would move His tag to the opposite terminus.

It is possible that the His tag is no longer visible (steric hinderance) after it binds protein B it might not bind nickel beads. If you just do a full cell lysate can you see your Histag protein when you express protein B? also, is the his tag n or terminal?

Yes, that's quite possible. I'm assuming you are running WB with anti-His AB, right? First, I would run WB with the unfolded flow-thought fraction of sample B from the affinity column. Second, try to unfold (destroy the complexes or even denature a bit your protein) the sample B BEFORE loading to Ni-NTA (or whatever you have for His-tag) column. And the last suggestion, I would move His tag to the opposite terminus.

Hi David,

I suppose protein B can just sticks to the NiNTA resin because of many reasons. I would suggest to use different tag eg. GST or use co-immunoprecipitation (CoIP). Protein B can also bind to different protein which also interacts with the resin so that is why it is visible. Regarding to the CoIP - if there is a possibility, use antibody against your protein A (tagged), then use agarose beads with proteins A and G to bind the antibody-protein complex and all other proteins complexed with your bait protein. Problem might be when antibody's recognition site is involved in protein binding.

Best regards

Dawid

Unlikely as the his tag would get in the way

Media that cells are grown in can prevent binding to His-Tag resin from working. I think there is a reducing agent or something but this is a well known phenomenon (look up "no binding to His-affinity resin when purifying from proteins expressed in cultured cells"). Also remove any DTT, mercaptoethanol, histadine, or chelating agents EGTA EDTA ect.

I recommend that you pre-clear protein B with His-tag affinity resin before you begin.

In general GST-pull down is preferable because His affinity resin also has ion exchange properties that make it more likely to randomly bind proteins.

Good Luck

I guess it can be the issue of transfection efficiency. When there is too much plasmid used, the efficiency will be low. Did you balance the transfection with single gene (tag or no tag) with empty vector such as pcDNA3.1?

While I understand that you would not be in a position to reveal the identity of 'protein B' do let us know a bit about the nature of protein B. More specifically, what is its concentration in the extract and how many Ni-NTA sites are roughly available on your column. Whether B is a basic protein and what is its size? Please treat this as the Path exam that a patient (in this case, represented by your tagged protein) has to go through.

Hi David,

a question do you do the pull-down in native conditions or denaturing?

I guess by your question "So is it possible that when the two proteins interact the HIS tag is no more visible" that you are doing it under native conditions.

I will recomend you to do a SDS-PAGE gel before the pull-down to check whether the tagged protein is there or not, and if the tagged protein is in the crude extract I will further check in which fraction the protein is lost during the pulldown. As you said, it is possible that the His-tag is hidden between the two proteins when they interact.

To overcome the purification problem you could also run two lanes of a native gel in parallel, in the frist lane load an extract of cells expressing only the his-tagged protein and in the second lane an extract of cells expressing both. Then immunoblot and check with the His-tag antibody whether the two lanes have run differently (if interacting, the band should run less in the second lane), or if you have antibodies against both proteins then check whether the antibody signal is localized in the same band.

I hope it helps!

am assuming that you checked your input material (by Western) to make sure that your tagged protein is appropriately expressed when you co-transfect it with protein B. If that is the case but your pull-down result (with Ni-NTA) yields no tagged protein it is indeed possible that the tag is masked in the complex such that it is inaccessible to the Ni-NTA. However, this would indicate that (a) your interaction with protein B is very strong and (b) that your protein B is in excess in the co-transfected HEK cells. One way to further analyze this issue would be to use different plasmid ratios for co-transfection (e.g., 5:1 or 10:1 of His-tagged:protein B) to see if you can now pull down some of the tagged protein. If the tag is masked by the interaction with protein B, you may still see no protein B, however. Ideally, you could do a reverse pull-down with an antibody to protein B (or a tag on protein B), to see if any of the His-tagged protein comes down. Another option to check if the His tag is masked by the interaction would be to either use a different tag or to put the tag in a different position (e.g., if it is now on the N-terminus switch to the C-terminus). It may come down to playing with the type and position of the tag on your protein(s). Masking of a His-tag (or conformational change such that it is no longer properly exposed for interaction with the NI-NTA) is definitely a possibility that could explain your initial result.

Depending on what pathways your proteins are affecting, is it possible that you see only Protein B, since co-transfection with Protein A is leading to cell death (Protein A by itself may not be causing cell death) and loss of a "pull-downable" complex?

Prior responses have covered technical details, but maybe something else to think about?

Hi,

i also observed a similar effect sometime. Be warry that interaction could mask the the his-tags. Try to make a native page and then a WB using a anti-His; probably you'll not see the his-tag as i experienced in the past!

Good luck!

Ouuuuah so many answers!! Thank you!

Ok so first of all I'm new with pull-down assays so I haven't probably done all the necessary controls. I know that my transfection protocol is efficient and optimized for transfecting my two proteins into HEK cells and cotransfection has no deleterious effect on the cells.

What I can tell you is that I first lyse my cells using a buffer with 1% triton (no sonication, no freeze/thaw) and I incubate the mix for 45 minutes on a rotating wheel at 4°C. Then, for the pull-down I don't use column to purify my proteins, instead I centrifuge my samples at 1000rpm for 2 minutes and I remove the supernatant then I wash the beads three times using a buffer containing 0.5% tween. For the final step I remove the supernatant and I add sample buffer 2x (Mercapotethanol) and I warm the sample for 10 minutes at 70°C.

I suppose that triton in my lysis buffer is not enough to break down the interaction between the two proteins so the pull-down is performed under native conditions. As I use SDS and mercaptoethanol in sample buffer, the western blot is performed under denaturing conditions, thus any shift in the protein weight can't be observed.

So after reading all your suggestion, I will perform the same experiment again but I will collect some protein sample in the crude extract and after the pull down. Is it right? If my theory is correct I should observe a huge amount of proteins in the "after pull-down" sample?

Edit: One thing is not in accordance with the fact that HIS-tag could be hidden by protein B: when I perform immunocytochemistry using the HIS tag antibody, I can clearly observe the tagged protein even when it's cotransfected with my protein B. I know that immunocytochemistry and pull-down are much different but I'm not sure this is the good reason!

I think you did not include chelating reagent such as EDTA in your lysis buffer, correct?

Yes it's correct!

What is the importance of EDTA in the lysis buffer? (I mean in pull-down assays)

People usually include EDTA in the buffer probably as inhibitor of the metallo-proteases to prevent protein degradation. But in His pull-down, you should avoid using high concentration of EDTA. I ever accidently used 1 mM EDTA. It did not interfere binding of 6xHis-tagged protein to Ni-NTA column. Maybe you can try 0.1 mM EDTA in your buffer. I know some people using that in purification of His-tagged proteins.

David, have you thought about using MicroScale Thermophoresis (MST) for investigating your interaction? MST would not only tell you IF your proteins interact, but you can also determine the binding affinity. The other big advantage is that this method is completely immobilzation-free and you do not need any tags for detection. However, you could run your experiment also in cell lysate in a purification-free setup if you are expressing your protein as a fluorescent fusion, such as with GFP.

You will find more information here

http://www.nanotemper-technologies.com/technology/

but please also feel free to contact me directly via [email protected] for further questions or to discuss suitable assay design.