You have to remember that when we work with proteins, we work with many copies of each kind of protein. As a result, the collection of proteins of any given size tend to move through the gel at the same rate, even if they do not take exactly the same tunnels to get through. Back to our analogy of the forest... If we were in a hot air ballon above the forest and watched 100 children, 100 teenagers, and 100 large adults running through the forest, we would see collection (or band) of children moving quickly, a band of teenagers moving slower, and a third band made of adults plodding their way through the forest. Likewise, proteins tend to move through a gel in bunches, or bands, since there are so many copies of each protein and they are al the same size. When running an SDS-PAGE, we never let the proteins electrophorese (run) so long that they actually reach the other side of the gel. We turn off the current and then stain the proteins (until we stain them, they are colorless and thus invisible) and see how far they moved through the gel. Figure 5 shows a cartoon gel and figre 6 shows a real one. Notice that the actual bands are equal in size, but the proteins within each band are of different sizes.
Figure 5. This shows a top view of an SDS PAGE after the current has been on for a while (positive pole at the bottom) and then turned off. The gel (gray box) has five numbered lanes where five different samples of proteins (many copies of each kind of protein) were applied to the gel. (Lane 1, molecular weight standards of known sizes; Lane 2, a mixture of three proteins of different sizes with a being the biggest and c being the smallest protein; Lane 3, protein a by itself; Lane 4, protein b by itself; Lane 5 protein c by itself.) Notice that each group of the three proteins migrated the same distance in the gel whether they were with other proteins (lane 2) or not (lanes 3-5). The molecular weight standards are used to measure the relative sizes of the unknow proteins (a, b, and c).
Figure 6. This photo shows a variety of different proteins being separated on a gel. This particular image is showing a serial dilution of the same protein sample to indicate how little protein is needed (16 picograms = 16 . 10 -12 grams) in order to be detected.
This image was taken from a home page operated by Hitachi Software (http://www.hitachi-soft.com/hitsoft/gs/fmbio/feb.htm)
There is a caveot to this method that you must always keep in mind. SDS-PAGE separates proteins based on their primary structure of size but not amino acid sequence. Therefore, if we had many copies of two different proteins that were both 500 amino acids long, they would travel together through the gel in a mixed band. As a result, we would not be able to use SDS-PAGE to separate these two proteins of the same molecular weight from each other.
