Scientists who routinely analyze proteins turn to SDS-PAGE, a proven method for separating proteins and peptides based on their molecular weight. First reported in the literature in the early 1970’s, this method has revolutionized biochemistry and also accelerated protein research. Proteins are denatured in SDS and reducing agents and separated in an acrylamide gel matrix upon applying an electrical current. The technology can also be used to assess the purity and relative abundance of a protein.
While running proteins on SDS gels is a good place to start, many researchers actually draw conclusions based on assumptions that they may not even realize they are making. Here are three common assumptions that researchers often make when drawing conclusions from their SDS-PAGE results.
Assumption #1: Expected band size means you have your protein
When you lyse a cell, you are extracting all the proteins present. Unless you have done some sort of purification step(s), many proteins present will have similar molecular weights. Therefore, identifying a band of the expected size doesn’t mean you have your protein. It just means you have a protein of that relative weight. If you need a positive proof of identity, a western blot must be carried out, which adds more steps and time to the traditional SDS-PAGE.
Assumption #2: Band intensity correlates to the amount of protein
A common quantification mistake is making the assumption that band intensity always correlates to the amount of protein. However, depending on whether you use silver or the classic Coomassie stain, staining intensity can actually widely vary for different proteins. Therefore, it’s not advisable to use staining for quantification purposes unless known quantities of a protein of interest are included on the same gel to calibrate staining intensity.
Assumption #3: Bands of an unexpected size correspond to contaminant proteins
Additional bands may not always reflect the presence of proteins other than the one you’re interested in. Instead, they may correspond to a different species of it. For example, smaller bands could be the result of breakdown products and larger bands may correspond to translational variants of your protein.
SDS-PAGE will always be the go-to method scientists use to study their proteins. While the nature of science is to continuously evolve, maybe it’s time researchers consider alternatives to this 40 plus-year-old technology. To get more complete information about your protein, including its presence, purity, concentration, similarity to another back and its functionality in a short amount of time than traditional SDS-PAGE, consider Tycho and leave no room for assumptions.