Hi.,

It is possible in case of membrane protein or proteins having hydrophobic patches . Try increasing the conc. of betaME hope it will work, further for identification of the protein u can do peptide mass fingerprinting by Mass spectrometry or do the western blotting using desired antibody.

For some proteins, for instance for some G protein coupled receptors dimers may be difficult to disrupt. betamercapto ethanol would not help much in this example, neither heating at high temperature will be useful. Try long term mild heating (40 degrees overnight) and at relatively normal salt concentration, i.e. do not add a lot of salt to try to force "dissociation".  Good luck

Hi there,

You may use loading buffer containing 8M urea in order to ensure optimal denaturation. The drawback is that every lane of the gel has to be laded with the buffer to avoid lateral diffusion of the samples during the running.

Thanks to all of you for your valuable suggestions and time, i will try each protocol, actually i am dealing with the protein whose function is still unknown and is conserved protein, so nothing is known about this proten weather it is memberane protein or something else. with the prediction tools which i have used it is seems to be memberane protein.

HI all,

even after trying with 8 M Urea i am getting dimer band in Induced one but not monomer band in any of the samples, is it possible that protein can only exsist in dimer form instead of monomer form, more over my protein is aspargine rich not histidine rich, so is it possible that long stretc of aspargine aminoacid is responsible for dimer formation.

Hello,  I think your protein may be annealing back after you reduce it. So you need to alkylate the reduced sulfite bonds.  Several companies  "carry reduction-alkylation kites .  Example Bio-Rad Cat #163-2090 or you can do it yourself.  It is a good practice to alkylate your samples every time you reduce them. Good luck.

Hi Isha

how are you expressing your protein? what am thinking of is maybe post-translational modification is happening like glycosylation which can affect how your protein migrates in SDS page which can lead to a higher molecular wt.  

You can check your protein using also non-denaturing PAGE, using lower percentage of SDS without using reducing agents such as DTT or BME, nor heating.  Then you can compare both apparent molecular weights, other way around may give some clues.

I agree with the possibility of glycosylation greatly increasing the MW. Working the exosomal marker protein CD63, the unmodified protein runs at about 25 kDa while the fully glycosylated form runs at about 60kDa.

Hi, Isha, have you settled down the problem? since I met the same problem with you.

Hi Su,

unfortunately, my problem was remain unsolved i tried lot many things but nothing worked with protein expression.At the mRNA level the protein was expressing but at the protein level we could not find the band of protein at the expected size, neither as dimer nor as single band. later on when we analysed bioinformatically we found that the protein was intrinsically disordered, so may be that could be the reason which may be inhibiting the protein expression

Hi everybody,

Yesterday I have done Western blot to my artificially created peotein which is ~7 kDa. Duribg sds-page I was notice there is a protein induces at the same time with my target protein. When I see this, I have done multiple IPTG (0,0.25,0.5,1 mM) induction. Whatever, finally I do western blot using Mab Antihis antibody, I see two different band. One of them 7 kDa, the other 14kDa and each of them gives same intensity at tge same IPTG concentration. I was planing purification and testing this protein . How to get rid of this situation. Is it really can be dimerization or another phenomenon.