Why is it important to equalize the charge density of the proteins by using SDS in the determination of molecular weight?
Hi there,
I'm not entirely sure what you want to know. From what I can gather, you are asking why you use SDS to equalize the charge when running protein through a gel?
If so, SDS confers a negative charge to all proteins. By doing so you make sure that the proteins are only separated by size. On a native gel, you would get separation based on MW, shape and charge. Some proteins would be negative, some positive and some neutral. To avoid this and get true MWs (at least of the individual subunits that remain when you denature your protein) you force 2 of the 3 factors into something you know (ie, shape is all linear, charge all negative). The only thing left then is molecular weight which you can now read off using your MW marker.
Hope that helped.
Hi there,
I'm not entirely sure what you want to know. From what I can gather, you are asking why you use SDS to equalize the charge when running protein through a gel?
If so, SDS confers a negative charge to all proteins. By doing so you make sure that the proteins are only separated by size. On a native gel, you would get separation based on MW, shape and charge. Some proteins would be negative, some positive and some neutral. To avoid this and get true MWs (at least of the individual subunits that remain when you denature your protein) you force 2 of the 3 factors into something you know (ie, shape is all linear, charge all negative). The only thing left then is molecular weight which you can now read off using your MW marker.
Hope that helped.
Sds binds protein, as a function of number of peptide linkages present. U can look up the literature for ratio. As a result, all proteins will have nearly equal charge/mass ratio. Hence resolution occurs on the basis of mass. Relative mobility vs log mass grap can be used to calculate the mass of ur protein. Hope it answers.
It's important to equalize the charge density to negate other physicochemical properties of the proteins (noted by Erik) from influencing the estimation of molecular weight. Since the SDS interacts with most (not all) proteins in a roughly equal mass/charge ratio, this allows you to estimate molecular mass based on relative mobility, with smaller proteins migrating further in a given time than larger proteins when subject to electrophoresis. Note that this does not work on ALL proteins, as there are some with particularly high ratios or clusters of charged amino acids that appear to exclude SDS from binding efficiently. Due to the inefficient binding, these proteins migrate much higher on the gel than expected, giving artificially higher molecular weight estimations by SDS-PAGE than they actually are. The best example of this I can think of is caldesmon.
And after all, the smaller the protein, the faster its migration from one side of the gel to the other!
Still, if I want accurate results, I will not rely on SDS PAGE... It is more qualitative than being quantitative.
The velocity of compound on electrophoresis depends on three things
1) size
2) charge
3) shape
Thus if you want to compare proteins by their charge, you have to make charge and shape as equal as possible. All the proteins have different charge, some may be positive, some negative, some may be elektroneutral (if the pH = pI). By adding SDS, you will denature them which causes loss of 3D structure and the shape is unified. Similarly, because approx. 2 molecules of SDS bind per 1 amino acid, the protein has the same charge per weight (i.e. per one amino acid).
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