2810712 Posted February 9, 2006 Share Posted February 9, 2006 SDS adds to mass as well as charge of the protein , to have the separation only on the basis of molecular wt. after adding SDS the q/m of all prots should be equal and as SDS also adds to molec. wt. of prot also, we should be able to get the original molecular wt. by comparing its mobility with that of a known sample molecular wt. In SDS PAGE we use sodium dodecyl sufate to make the ratios of q/m s of two diff peptides nearly equal still keeping the difference between them the same. This tells that this ration is only important here... why??? I think the difference in the displacements of the two peps in the gel depends only of the ration of their q/ms. What is its expression??? DON'T mind Yggdrasil, if u r here... Shrei Link to comment Share on other sites More sharing options...
Nashyboyo Posted February 10, 2006 Share Posted February 10, 2006 The mass/charge ratio is what separates proteins of different sizes. I can't think of the formula. The bigger the protein the more SDS associates with it thus more charge. More charge gives more mobility towards the anode and away from the cathode. Link to comment Share on other sites More sharing options...
2810712 Posted February 10, 2006 Author Share Posted February 10, 2006 thnx for reply we can't make definite statements about mol.wt.s of proteins without a formula... hrushikesh Link to comment Share on other sites More sharing options...
Bluenoise Posted February 10, 2006 Share Posted February 10, 2006 we can't make definite statements about mol.wt.s of proteins without a formula...hrushikesh You also can't make one with a formula. Electrophoresis is a very variable form of measurement. Each run will go sligtly different due to the many factors involved that you really can't account for. I'll give you the formula, but I don't really care to explain it, since it's fairly useless anyways. ZeE = fu The only way to make a real comparison is to run a set of proteins of standard weight along side your samples. Then estimate. Alternatively you could plot the distance vs time fit it to this formula. However it is ONLY good for samples run on the same gel at the same time. logM = a - bx M= mass of protein. x= distance travelled a,b are constants individual to every gel. enjoy. Link to comment Share on other sites More sharing options...
Nashyboyo Posted February 11, 2006 Share Posted February 11, 2006 Yeah that's right. You have to construct a calibration curve using the ladder to estimate the Mr of the protein(s). Link to comment Share on other sites More sharing options...
2810712 Posted February 12, 2006 Author Share Posted February 12, 2006 Thanks for those formulae...today its fairly hard to understand and estimate the errors so as to get correct forumula... and as the errors are variable with gels it seems fairly useless. hrushikesh Link to comment Share on other sites More sharing options...
Yggdrasil Posted February 12, 2006 Share Posted February 12, 2006 In order to get an accurate molecular weight determination of a protein, you generally will not rely on only one technique. Proteins will often run at a different location on the gel than predicted for various reasons, so molecular weight calculation from SDS-PAGE can often be misleading. However, in addition to SDS-PAGE which measures the molecular weight of the denatured protein, techniques such as analytical gel filtration or analytical ultracentrifugation are used to measure the molecular mass of your protein in its native state, and can provide independent confirmation of the molecular mass determined using SDS-PAGE. However, if you just need a quick confirmation that your protein is of the correct size, SDS-PAGE provides a reasonable estimate of molecular weight. Link to comment Share on other sites More sharing options...
Bluenoise Posted February 12, 2006 Share Posted February 12, 2006 Or as always when in doubt just do mass spec. Though you should have a very pure sample Link to comment Share on other sites More sharing options...
2810712 Posted February 16, 2006 Author Share Posted February 16, 2006 that helped frndz, informative... thanks alot. hrushikesh Link to comment Share on other sites More sharing options...
Run Posted March 5, 2006 Share Posted March 5, 2006 The separating difference by SDS-PAGE is because of friction effects. the proteins are separated in a mass dependent manner. If a constant force (F) would be applied on the proteins the low molecular mass (m) proteins would have a higher acceleration (a = F/m) compared to the high molecular mass proteins. However, not only the acceleration but also the force applied on the SDS coated proteins is mass dependent, since high molecular mass proteins carry more negative charged SDS (sodium dodecyl sulfate) (ratio 1.4:1). This results in a same velocity for all the different proteins. However, counting for the friction effects contributed by the gel, the heavy proteins are delayed more compared to the small proteins resulting in a mass dependent separation. Link to comment Share on other sites More sharing options...
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