I just made a SDS-PAGE with a top layer of stacking gel and a bottom layer of separating gel with different pH values of 0.5M Tris-HCl. The stacking was 6.8 and the separating gel was 8.8. What about this pH change makes the gels different? What effect does this have on the proteins that I ran?
The stacking gel concentrates proteins loaded into the sample wells so that they are resolved as a unified "line" once they enter the stacking gel. The reason for the lower pH is that this "lower ionic strength implies higher electrical resistance and consequently a higher electric field, provoking the faster movement of the proteins and of every other charged particle in the gel. Such a high electric field coupled with the glycine in the running buffer (that will not go into the resolving gel due to the pH), helps to clean the sample from the Cl- ions from the Tris-HCl buffer. That concludes in a stack of clean, denatured, and equally charged proteins in the boundary with the resolving gel; this will account for a good quality PAGE."
Bassically, in the stacking gel the pore size is larger so it gives a chance for the larger proteins and smaller proteins to equalize with each other.
"As the proteins are denatured and linearized by heating, SDS and dithiothreitol (if there is any di-sulfide bond), the proteins are loaded onto the wells on the stacking gel. The denatured proteins have a uniform mass to negative charge ratio. Since the current run from negative (top) to positive (bottom), the proteins move toward the bottom. When the electricity is turned on, the proteins and Tris-glycine enter the stacking gel. In stacking gel with pH 6.8, the N-terminal amino group of the proteins and amino acids are protonated at equilibrium which makes them less negative. The average electrophoretic mobility is very slow. A Gly-chloride ion boundary is formed since glycine moves slower than chloride ion. However, glycine still runs slightly faster than other proteins. As a result, the glycine keeps pushing the protein towards the chloride ion. In other words, the proteins are trapped between glycine and chloride ion. The proteins form a very tight band inside the stacking gel.
Once the protein reaches the resolving gel, the pH changes from 6.8 to 8.8 and the pores are smaller. As pH increases, the N-terminal amino groups are deprotonated. Amino acids and proteins are more negatively charged at equilibrium than in stacking gel. As a result, glycine moves faster than proteins. Glycine and chloride ions move ahead of the proteins. As the pores are smaller in the resolving gel, the size of the protein determines the mobility (speed). So the smaller the protein is, the faster it will reach the bottom."
The other major difference between the two is the amount of acrylamide in the upper (stacking) gel - it's generally around 4%, while the lower (resolving) gel can vary from 6 or 8% to 20%, depending on the size of the protein(s) you're looking for.
When you load your samples in the wells at the top of the gel, then start the current, not all of the sample enters the gel at the same time. The purpose of the stacking gel, with its lower pH and low acrylamide percentage, is to "stack" all of the proteins in your sample into as narrow of a band as possible, so that they all enter the resolving gel at essentially the same time. If you didn't have that stacking gel, your band(s) of interest could be very diffuse, and may not match up with your molecular weight marker.