[JanetteB]

My biochem professor is requiring us to research and present on the theory behind SDS-PAGE, and I need some help with the details.  I've done a lot of research, but apparently I'm missing some key concepts, so I was hoping someone might be able to help me fill in the gaps or refer me to some additional resources.  I need help with these concepts:

1.  Role of SDS- from my understanding, SDS denatures the protein sample to its primary structure and coats the protein uniformly with negative charge.  This equalizes the charge-to-mass ratio of the proteins so that separation is related to size only, not charge.  I've been told to explain this further, and to think about the fact that a larger protein (which should move slower) will have a larger negative charge from SDS (more negative should move faster?).  Not sure how to reconcile these two ideas, unless the relative charges on each protein moves them forward by the exact same amount.  What am I missing here?

2.  Purpose of stacking gel- I thought the purpose was to pack the proteins into a tight band between the chloride and glycine ions, so that all the proteins would enter the resolving gel simultaneously, and so that better resolution would be acheived in the resolving gel.  My professor says I'm missing the main purpose of stacking, however, nothing I've read seems to contain any other information.  Help please!

3.  How stacking occurs- My understanding is that the chloride ions move at the solvent front because of their smaller size and high negative charge, and the glycine ions are retained longer because at a pH of 6.8 they are mostly protonated, and thus neutral.  The proteins then move between the chloride and glycine because they are larger than chloride ions, but more negatively charged (from the sds) than the protonated glycine.  Once again, I've been told I'm missing some details, but can't figure out what.

I appreciate any help/information anyone can give me, thanks!

[Wreath]

1) Bigger proteins are moving faster than smaller. When you run SDS-PAGE, you have bigger proteins on the bottom of the gel. You're probably confusing with electrophoresis in concentration gradient, where the pores of PAA or agarose are getting smaller and smaller. In standard SDS-PAGE there are pores with the same size so the more negative proteins could go faster...

Sorry, didn't have time to look the 2 and 3, I'll do later :-)

[lipox_1]

The first reply was not correct!
Big proteins are slower, small proteins are faster!

[Wreath]

Yep, sorry, I realized later I was kinda drunk or something blew my mind, but there were no other answers so I left it. Sorry again, that really wasn't correct.
Well, I've been reading a little. When SDS connects protein, it binds in constant ratio 1,4g SDS / 1 g protein. This nonspecific binding interaction yields species with constant charge per unit weight (from the sulfate groups of SDS), since the contribution from charged protein side chains becomes negligible. Migration distances are then influenced only by sieving and are directly related to the molecular weight of the protein or subunit. Better answer for you? 

[diablo]

ad 3)

More details about the initial events upon the application of the electric field and events in the stacking phase

The sample is routinely loaded in a volume that results in a sample extension of roughly 0.5-1.0 cm in the direction of the electric filed.  To decrease this sample spread before the micelles enter the resolving gel, the protein micelles are stacked.  Stacking occurs in the top region of the gel (the stacking gel)


which has a larger pore size than the lower region (the resolving gel).   The larger pores of the stacking gel don't inhibit migration of large proteins much.  This actually helps in the process of stacking.



Stacking occurs as a result of the differential rate of migration of the protein-micelles in the presence and absence of chloride ion "clouds" that initially surround and shield the SDS/protein micelles.  To achieve this clearing of the chloride cloud, the titrate-ability of the glycine anion is employed.  When the electric field is turned on, glycine, in the running buffer at pH 8.3 is slightly negatively charged and as such it carries the current in the buffer until it enters the sample buffer, pH 6.8, where the glycine becomes neutral as the amino group becomes totally protonated and the carboxyl group remains de-protonated. 



The chloride ions in the sample buffer and the gels create a "cloud" through which the SDS/proteins micelles can migrate only relatively slowly in the electric field. It is as if the chloride ions shield the micelle strings from experiencing the full force of the applied electric field (they don't move very fast).  However, the chloride ions in the sample buffer and the gel buffers carry the current in these parts of the system initially, and start their migration toward the positive electrode upon the application of the electric field.  As the fastest moving species in the mix, the chloride ions, clear from the top of the sample buffer moving toward the positive electrode at the bottom of the gel, the slower moving SDS/protein micelles are left "out of the cloud of chloride".  The entering glycine changes from negatively charged to neutral upon entering the pH 6.8 environment, leaving the protein-micelles unshielded so they now move faster toward the positive electrode than the micelles still in the chloride cloud lower in the sample buffer or in the gel.  By the time the loaded sample reaches the resolving gel, the protein-micelles have managed to form a nice tight band < 1mm wide.  This accounts for one component of the stacking phenomenon.  The other components result from the slowing of the

original source: http://sdspage.homestead.com/

greets diabloo

[mlinke]

Do we have the same professor? 

I have a related question. When SDS binds to a protein, it denatures is. If we proceed to do Western blot what kind of antibodies do we use? I don't understand this, how does an antibody recognise this denatured structure? I have been looking at cathode and anode buffers used in Western. The only difference from SDS-PAGE is the lack of SDS and presence of methanol. What does methanol do? Does it take some SDS of the proteins?

[JGK]

http://www.molecularstation.com/sds-page-gel-electrophoresis/