[2810713]

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???

Shrei

[Yggdrasil]

In SDS-PAGE, the SDS molecules coat the polypeptide.  This is important for two reasons:
1) the negative charge of the SDS masks the natural charge of the polypeptide and gives all polypeptides the same charge per mass ratio. Since the charge-to-mass ratio for all polypeptides is the same, they will be equally attracted toward the positive electrode.  
2) the SDS denatures the polypeptides so that all the polypeptides are linear.  This prevents the shape and packing of the polypeptides from becomming a factor in their migration through the gel.

As a result, migration of the peptides through the polyacrylamide gel is dependent on only the lenght of the polypeptide strand.  Longer fragments will encounter more resistance while passing through the gel, while smaller fragments can more easily travelse the gel.  The length of the polypeptide strand is roughly proportional to its molecular weight, so SDS-PAGE is useful for molecular weight determination.

When analyzing the results of an SDS-PAGE analysis, the distance traveled through the gel is linearly proportional to the log of the molecular weight of the polypeptide.

[2810713]

Thanks alot.
Now one q. reamins- How does SDS do that - i.e. enev if proteins have different AA sequences how  does it make charge/mass approx. equal

Shrei

[Yggdrasil]

Sodium dodecyl sulfate (SDS) is an anionic detergent which is amphipatic, meaning that it has both hydrophobic and hydrophilic regions.  Like most detergents, it will bind to large molecules in solution and coat the molecule.  In household detergents, this property allows them to solvate oils and grease so that they can be more easily removed from dishes and clothes.  In SDS-PAGE, the SDS can interact with hydrophobic residues using its nonpolar tail and it interacts with hydrophilic residues through its charged head.

Since SDS coats the protein pretty much equally along its lenght, the amount of charge (determined by the amount of SDS, which is negatively charged) is determined by the length of the protein (which is roughly proportional to its moleuclar weight).  The amount of SDS which binds to the protein is great enough to make the native charge on the protein negligible.

[2810713]

That really helped alot.  
Again- if q1 is original charge on that protein and the added by SDS charge is Q1 and M1 is protein molec.wt. and m1 is the toatl molec. wt. of SDSs bound to it. Then the new charge/mass ration is
q1+Q1/m1+M1   for some other protein it is q2+ Q2/M2+m2 . Now i' just confused about what we actually mean by approximately equal...
Is the difference between these q/ms is made very less or the ration of them is made nearly equal. I realised that these two are diff. things when I saw the expression for 'work done in isothermal reversible process' as a function of initial and final volume. The ammount of work done only depends upon their ration i.e. even if the difference between the initial and final states is different, same work may have been done to achieve that change!!!
So, i thought some such kind of 'biased' function might be guiding this process. Is it there???

By the way you are really helping me alot.Thanks.


Shrei