When I'm preparing the samples, after added the SDS-PAGE, the protocol sends to heating these samples at 95°C for 10 minutes. My doubt it is about the integrity of the protein. When I heat the samples at this temperature I denature it, so the protein loose its quaternary/tertiary structure. But when I heat it this procedure can be sufficient to break a disulfide bond that link its two subunits? In the case of exist only one disulfide bond that link these two subunits. Is it tells us that if appear two bands instead of only one band, this effectively happens? I mean is it possible that one antibody made for an entire protein (with these two subunits) left to mark this protein, to mark these two subunits separetely? Is it possible?
As indicated in the previous answers you need to have a reducing agent in the SDS-PAGE sample buffer to cleave the disulphide bridge. DTT works well and is much more powerful than 2-mercaptoethanol: a final concentration of DT in the range 50 to 100mM is typical. DTT solutions are not stable and it should be made freshly or stored frozen. You can also use the more powerful reducing agent TCEP (tris(2-carboxyethyl)phosphine).
With regards to the antibody reaction. If you are using a monoclonal antibody then it may react with either one other subunit or both. If both subunits are similar then they may each have an epitope recognised by a single monoclonal antibody. It is also possible that the structure of the epitope requires both subunits to be bound together to form the structure recognised by the antibody. In this case, after PAGE separation, there may be no binding to either subunit. If you have polyclonal antibody then it is likely ( but not certain) that there will be binding to both subunits after they have been separated by PAGE.
When single cysteine is present in single subunit, which mediate dimerization of the potein, leads to the appearance of two bands in the SDS-PAGE. Heating of sample upto 95 degress will not break this covalent bond. When we see two bands in the gel, that means the oligomeric status in the solution exists in equilibrium between dimer and monomeric species. Two bands in the SDS-PAGE could also be because of the DTT in the loading dye which will convert the dimeric form into monomeric form.
There is one way of concluding this is that if you will treat your sample with 8 mM of B-mercaptethanol, all the dimer will convert into monomer, however if you will add copper phenathroline plus copper sulphate into the sample all the monomer will convert into the dimer.
And yes you can use the antibody which is generated against single subunit to detect two bands corresponds to monomer and dimer in western blot.
Hope this will work.
Nice answer by Manoj, I just want to add that sometimes SDS stabilizes the dimer, So you can boil the samples for longer time (20 min). Protein may come as monomer.
Thanks for your answer Manoj. So the band that I must to see have between 83 and 92 KDa, occur that I can't see a band at this weight, but I'm seeing two bands that I think it is the two subunits, because these two subunits the alpha has 69 KDa and the beta has 34 KDa, that match with the bands that I'm seeing.
Besides that, I know that these samples must have this protein because its gene expression. In regard of the antibody, is it possible that it detect the two subunits, even that they are heterodimerics?
If you think the dimerization is mediated by cysteine only, then incubate these protein subunits in the presence of 5 mM 1, 10 phenathroline and 1.5 mM copper sulfate at 20 degress for 24 hours. After SDS-PAGE, you should see your expected size of heterodimer i.e as mentioned by you between 83 and 92 kDa.
If the antibody you are using is polyclonal and raised against the complete heterodimer, then you can probe both the subunits in western.
Heating step is normally with reductive reagent, which can reduce most if not all, s-s bonds, sending any protein into a single band. However, proteins often have modification. It is often that you see a double band from a single protein by western. The bands may also come from protein truncation/degradation.
Thank you for your answer Ziguo Zhang. In the case of this particular protein, there is only one dissulfide bond that link two subunits. Is it possible to be degradation if the beta actin is showing a weak band?
Please try to contact Professor Yves Combarnous on research gate, He is an expert in Disulfide bonds and the behavior of protein subunits. He certainly have all the answers you need concerning this subject.
As indicated in the previous answers you need to have a reducing agent in the SDS-PAGE sample buffer to cleave the disulphide bridge. DTT works well and is much more powerful than 2-mercaptoethanol: a final concentration of DT in the range 50 to 100mM is typical. DTT solutions are not stable and it should be made freshly or stored frozen. You can also use the more powerful reducing agent TCEP (tris(2-carboxyethyl)phosphine).
With regards to the antibody reaction. If you are using a monoclonal antibody then it may react with either one other subunit or both. If both subunits are similar then they may each have an epitope recognised by a single monoclonal antibody. It is also possible that the structure of the epitope requires both subunits to be bound together to form the structure recognised by the antibody. In this case, after PAGE separation, there may be no binding to either subunit. If you have polyclonal antibody then it is likely ( but not certain) that there will be binding to both subunits after they have been separated by PAGE.
Thank you for your answer Professor Jackson. Well, in fact the antibody that I used is polyclonal.
If I don't use any reducing agent, I can see one band instead of two? Or don't have this possibility to not use reducing agent?
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