Hi Chhaya: even the most powerful urea or SDS PAGE will not separate two proteins with very similar MWs. In such cases try to use another protein feature, such as the electric charge. I would suggest to perform a first separation based on the two proteins different net charge by IEF and then proceed to a second separation by mass by means of SDS-PAGE. Thus, I strongly recommend you to consider 2-DE for your research. 

best regards

I used gradient reverse phase HPLC to separate a hormone (Oxytocin) at 1007 amu from its related substance at 1009 amu.

In theory, a significant size difference (~ 10%)  is required to reliably separate proteins by PAGE. However, sometimes separations work in practice that shouldn't work in theory, for example the separation of Na/K-ATPase alpha-subunit isoforms using sodium tetradecylsulphate instead of SDS. Nobody knows why that works. 

To have any chance at all, make sure your buffers are selected for optimal band stacking in the stacking gel. Ornstein's 1962 paper (doi:10.1111/j.1749-6632.1964.tb14207.x) explains how this works, you may also consult Jovin's papers from 1973 (doi:10.1021/bi00729a014, doi:10.1021/bi00729a015, doi:10.1021/bi00729a016). 

Adapt the gel concentration to the size of your protein. Using shallow gradients can further improve resolution as it reduces band spreading by diffusion. Steep gradients, however, increase the size range of the gel at the expense of resolution. 

Acidic gels cannot be polymerised with APS/TEMED and are often polymerised using a Fenton system (Fe/H2O2 doi:10.1039/CT8946500899). Such gels are brittle and easily break during staining. Rabilloud et al. (1996, doi:10.1002/elps.1150170112) have published a photo-polymerisation system with methylene blue that results in more flexible gels.

Under acidic conditions, positively charged detergents may work better than SDS (or even lithium dodecylsulphate), I have published a set of methods with CTAB (Buxbaum 2003, doi:10.1016/S0003-2697(02)00639-5). 

Dear Chhaya:

I completely agree with comments from Barros-Velazquez. If  these techniques are not avaiblable for you, try to use SDS_PAGE gradient gels. Sometimes, separation of proteins showing similar Mrs improves using 4%-20% acrylamide gradients.

Dear all

Thanks a lot for your valuable suggestions; will explore all the possibilities especially IEF, SDS-PAGE gradient. Fenton system suggested by Engelbert is an excellent suggestion. Thanks for the papers sent

Thanks