I am doing pull down using His tagged protein A (28kDa) and an untagged protein B (47kDa). I incubate the proteins in presence and absence of a drug. In presence of drug protein A should interact to B. Otherwise there is little interaction of these proteins. What should be the result of this pull down? Should I see the pulled protein @ 47kDa using the specific antibody for B? That is, a better intense band compare to minus drug?
What I could see in the coomassie stained gel is, better band in + drug @ 47kDa area. Does that mean pull down worked? I also got something around ~75kDa (would this be the complex of protein A and B). I normally elute using 300mM imidazole and incubated @ 95 degree in SDS loading buffer. So the proteins might dissociate. I am waiting for the WB result soon. Please give your suggestions.
Unless the 2 proteins become attached by a covalent bond, such as a disulfide bond, they will appear as separate bands at 28 and 47 kDa on SDS-PAGE, not as a single band at 75 kDa. If you are not including a reducing agent in the experiment during the pull-down or during the sample prep for SDS-PAGE, it is possible that such a disulfide bond could form if there are free, surface exposed cysteine residues in both proteins. To test for this possibility, prepare the sample for SDS-PAGE with reducing agent.
It sounds like your pull-down experiment is working. You used an antibody against B and pulled down A with greater efficiency when the drug was added that causes A and B to bind more tightly.
Hi Lax Lee,
A few things that could mislead in pulldown assays are insufficient washing after your receptor on column is incubated with your ligand. there is always some non specific binding of your ligand/receptor to the column that can be minimized by including an optimized concentration of imidazole (in this case) in your binding buffer. The band at a mol weight of A+B is very interesting that could indicate the interaction between A and B either through disulphide bridges in absence of reducing agents in your buffers or some very strong hydrophobic interactions between A and B that survived incubation at 95 deg C in presence of SDS. It might sound remote but i personally have experience of some protein that formed dimers spontaneously and survived treatment with SDS at 95 deg C. Proper +/- controls for each of the condition might direct you in a way to conclude your results.
hope that helps
sincerely
Venu
You can please follow the link below:-
https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/pull-down-assays.html
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