Question

To prepare an SDS-PAGE gel, you have been provided with a stacking gel buffer (Tris-Glycine HCl, pH 8.8) and a resolving gel buffer (Tris-Glycine HCl, pH 6.8). After running the gel, you find that the bands are not of the expected molecular weight. Why may this be the case? What would you do to correct this, and why would you expect it to work?

Answer 1

the bands are not of the expected molecular weight

While running gel process, at ph 8.8 the glycine molecules are mostly negatively charged and can migrate much faster than the proteins. So the glycine front accelerates past the proteins, leaving them in the dust

because of this  proteins are dumped in a very narrow band band at the interface of the stacking and running gels and since the running gel has an increased acrylamide concentration, which slows the the movement of the proteins according to their size, the separation begins.

For correcting this

Gel wells are around 1cm deep and you generally need to substantially fill them to get enough protein onto the gel. So in the absence of a stacking gel, your sample would sit on top of the running gel, as a band of up to 1cm deep.

Rather than being lined up together and hitting the running gel together, this would mean that the proteins in your sample would all enter the running gel at different times, resulting in very smeared bands.

So the stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.