Do you know If the urea is quite more efficient than SDS as a protein denaturant? I always experience a smear in my SDS-PAGE and I thought Urea may help me in getting rid of this smear.
Hi Haitham,
Urea is usually added as a denaturant for nucleic acids. It breaks hydrogen bonds between base pairs allowing strands to separate. For proteins, I would stick with SDS which denatures secondary and some tertiary protein structures while conferring a negative charge. Have you tried reducing agents with your SDS-PAGE? Beta-mercaptoethanol or dithiothreitol can help further denature proteins by breaking disulphide linkages. This can help with some additional forms of protein folding that you may be experiencing.
Hi Haitham,
in my experience, if you cook your samples properly in Laemmli-buffer prior application to the SDS-PAGE and the composition of both, Laemmli and SDS-PAGE is correct, the SDS-PAGE method does not lead to the described smearing of the bands. So, putting in other words, if your lanes smear, you most probably have some proteases activ in your sample. You can avoid this by adding protease inhibitors (normally mixtures are commercially available) to the sample earliest possible in your preparation steps.
The urea gel I only used for those cases, where I needed a reversible denaturation of the proteins, or a separation of the nucleic acid oligomeres according to their molecular weight. In this context, don't forget that SDS has also the function of evenly loading the proteins with charge, so they will band only according to their molecular weight...
Hope this helped a bit.
Best regards,
Yilmaz
Yes Katrina for your answer, I figured it out, and yes sure I am using BME in my recipe.
Thanks a lot Dear Yilmaz,
Actually, I am using it the way we all use here in our lab, the problem is that the smear is not small size one but rather large size and the protein is a recombinant and glycosylated one it is a part of IgT (salmon).
Hi there,
In order to improve/optimize SDS mediated solubilization/denaturation of proteins, I sometimes use 8M urea in my SDS PAGE loading buffer. The results are excellent, the only thing to be careful with is that every wells of the gel have to be loaded with this urea/SDS loading buffer to avoid lateral diffusion of the samples during migration.
Glycosylated proteins often migrate as a smear on SDS-PAGE. Can't you try a brief treatment with EndoH or PNGase F before electrophoresis to see if this is the problem?
Thanks Alejandro, actually that what I was intending to do, the problem is that the MS analysis showed no Ig peptides at all.
What is the source of your proteins. Can you expect glycosylation. If you use Urea, prepare fresh to prevent Urea modifications of the proteins (http://www.ionsource.com/Card/carbam/mono0005.htm).
Much obliged dear Winfried,
My protein is a recombinant protein in a mammalian hek cell, I am expecting a glycosylation as it is a part of a unique Immunoglobulin in fish called IgT.
The smear you get is mostly because of very high concentrations of detergents in your sample. For checking in SDS you can quickly exchange the buffer via amicon filters and then run SDS. Do you calculate the concentration of protein before loading on the gel? maybe you are loading too much protein?
Thanks Farhan
If you mean the detergent in the sample loaded in the well, I am loading my sample (Diluted 5 times with the buffer containing SDS) from stock which is 1 mg/ml
Well, normally i donot have problems in using excess of the SDS dye, but what you can try is to dilute the sample in pbs or tris then try running the gel after adding dye. what could also help is that if your sample is having some debris, then after adding the dye and denaturing it, centrifuge the sample for 2 mins at max speed, then load the supernatant on the gel.
Best
Did you consider the salt concentration of your sample? Considering that you are purifying out the recombinant protein, most purification procedures of that nature require a good amount of salt to reduce non-specific binding. If you are only analysing it for characterization purposes, for running the gel, i suggest that you do an acetone precipitation first to desalt, and resuspend a 1X loading buffer for denaturing.
Large smears on a gel are mostly due to high salts, if it is a smear throughout the lane, chances are you have got DNA in your sample. Small glycoproteins should give you a small smear, depending on how many glycosylation sites there are.
Urea is different denaturant than SDS. Treating your sample with ~5 M Urea than adding about 3-5 folds access of sds/protein (w/w) + 5 min 95C will eliminate your smearing problem. If you have salty samples I suggest you first dialyzed tham 1 hour against water or very mild buffer than estimate what is your protein concentration and continue with the Urea SDS treatment than load the gel.
Good luck
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