I am doing SDS-gel non-reducing for protein sample. Sample preparation include mixing LDS sample buffer (similar to SDS) with protein sample in 1:4 ratio followed by heating at 80 degrees for 10 min. The protein appears with a monomer and dimer (formed from H-bonding of two monomers). From literature, it is confirmed that SDS breaks H-bond so in principle I should not see the dimeric form of protein. Does dimer appearance correspond to the amount of low LDS ratio used in sample preparation?
Looking forward for suggestions and please correct If I am wrong
Do you have the gel? There is still a lot left of dimeric form? Is your protein cytosolic or membrane? Despite heating and adding LDS buffer, it happens often to add normal quantities of Laemmli buffer and be still left with a very small amount of dimeric form, but yes, having not enough SDS (or LDS) can definitely cause that.
The protein is 91 % pure, purified from milk and purchased from sigma. it is cytosolic. Thank you for the reply.
Hi Khadija, I just want to impart my thoughts here in this situation.
In non-reducing PAGE, only it is the SDS, which causes denaturation or more precisely linearization of proteins. Factually, SDS breaks the H bonds, which are an important feature in protein stability of its secondary structures, but SDS not necessarily denature these secondary structures completely as often the non-covalent protein-protein interactions (dimers) are very stable and mostly in abundance as well so SDS can only linearize them more effectively under reducing PAGE conditions along with common chaotropes like Urea or Guanidine HCl, whereas alone SDS is not enough to dismantle these H bonds 100 percent. Also, heating has a role in H bonds disruption and yet you have seen the dimeric form. Which is the main question here.
If your protein is a hetero-dimer so only in reducing PAGE it should exhibit 2 close bands, so in my view there could be a chance that your protein has already undergone structural degradation and / or your running condition is not the proper non-reducing one, hence giving this technical artifact. It could be that your LDS already has included any reducing agent, which you are not well aware of. You should also compare your protein with a reducing PAGE to see whether there is a single band or more bands to get idea about your sample status. If your protein is homo-dimer so in all conditions (native or reduced) only a single intense band will be there, as they tend to overlap due to less weight and migrate same distances in the gel.
You can also use Aldrithiol-2, which is a free-thiol oxidizer, which keeps intramolecular disulfide bridges intact to avoid any artifact or flaw due to improper sample preparation and in the running of PAGE.
Regards....
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