Sodium Dodecyl-Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is a common tool biochemists use to find out the apparent molecular weights of proteins in a sample. The technique separates proteins by their sizes (or lengths of polypeptide chains).
In the procedure, the sample to be analyzed is mixed with SDS, an ionic detergent. SDS binds to all proteins and breaks up the weak (non-covalent) bonds of the proteins, "smoothing" them out so that they exist in long rope-like SDS-polypeptide micellular chains. Each SDS molecule has 1 negative charge. Also, SDS binds at a rate of 1.4g SDS per 1g Protein which gives all SDS-bound proteins roughly the same charge to mass ratio, and hence equal mobilities in an electric field.
The sample (SDS-protein solution) is placed on top of a polyacrylamide gel. The gel contains TrisCl buffer and is covered at the top and bottom with liquid Tris-glycine running buffer. An electric field (– to +) is applied across the gel from the top phase (stacking gel) to the bottom phase (resolving gel), causing the negatively charged "current-carrying" anions to migrate down through the gel toward the positive electrode.
There are three important "current-carrying" anions in SDS PAGE: 1.) glycine from the Tris-Glycine buffer found in the buffer above the stacking gel and below the resolving gel; 2.) chloride ions from the Tris-Cl buffer in the sample buffer, the stacking gel, and the resolving gel; and 3.) SDS/protein micelles. When the electric field is applied a "current-carrying" anion race through the gel is initiated.
The mobility of each "current-carrying" anion in the electric field is proportional to its charge/mass ratio; and the SDS-protein micelles all with the same charge/mass ratio are sieved by their size in the resolving gel matrix: small protein-micelles move the fastest while larger ones sieve more slowly through the gel matrix. After a set amount of time (usually an hour or two), the protein micelles will have differentially migrated through the gel with the smaller proteins traveling farther down through the gel than the larger ones, which remain closer to the top of the gel. Thus proteins may be separated roughly according to size (and therefore, molecular weight).
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)
Diagram of gel:
<-----------(Tris-Glycine pH 8.3 running
buffer above the stacking gel)
|.. .. .. ..|<-------(Proteins+SDS+TrisCl-
|.. .. .. ..| pH 6.8 in the loaded sample)
| X X X X|
| X X X X|<-------(TrisCl- pH 6.8 found
| X X X X| here in the stacking gel)
| XXXXXXXXXXXXX|<-------(TrisCl- pH 8.8 resolving gel)
<-----------(Tris-Glycine pH 8.3 running
buffer below the resolving gel)
(+)(+)(+)(+)(+) DIRECTION OF
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 micelles upon encountering the various buffer-gel interfaces.