How SDS-PAGE Separates Proteins

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Have you ever wondered how SDS-PAGE separates proteins? As the first critical first step in the Western blotting process, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins by their molecular weight. It acts as the crucial first step to successfully detecting proteins when you are Western blotting.

What's PAGE?

PAGE is an assay by which proteins migrate through a polyacrylamide gel matrix with the application of electric current. Because of the denaturing conditions and coat of negative charge provided by SDS, the proteins will migrate based on size almost exclusively (without influence from native charge or structure). To determine the size of the sample proteins, a molecular weight marker (also referred to as a protein ladder) containing proteins of various known sizes is run alongside samples on the same gel. Often, these ladders are pre-stained so the progress of the protein can be readily visualized. Proteins can then be identified by comparing their migration patterns to those of the molecular weight marker proteins. 

How does protein travel through the gel?

The protein ladder is a purchased reagent that undergoes quality control to ensure that it is indeed prepared properly and ready for use in SDS-PAGE; however, experimental samples must be prepared in the lab. The goal of sample preparation for Western blot is denaturation and uniform coating of the proteins with a negative charge. These steps allow the proteins to travel effectively through the gel matrix based exclusively on size.

During the electrophoresis step, the proteins travel through a gel matrix, inside a gel running system (like an Azure Aqua), where an electric current pushes proteins through the gel. The current shoves the proteins to equilibrium, where they won’t move anymore.

If either of these requirements fail, protein separation will not occur properly with SDS-PAGE. The protein ladder will, of course, be unaffected by other sample preparation and will migrate properly.

Loaded gel in electrophoresis for SDS-PAGE
Protein samples loaded into a gel inside an Azure Aqua Vertical Gel Running System

Common issues with SDS-PAGE

Unfortunately, appropriate ladder migration and separation does not guarantee the same of the sample proteins. Usually when there are migration issues with SDS-PAGE, we consider a number of factors such as buffers and gel quality. However, in this case, the successful migration and separation of the molecular weight marker indicates that those factors are working appropriately. This leads to the question of how are the protein samples differ from the protein ladder. 

Just getting started with Western blotting? This page has everything you need to prep for your first Western blot. Good luck! Use the form on this page to ask us any questions along the way. Talk to you soon in another post.

Troubleshooting resources for SDS-PAGE:

quantitative western blot basics


Get a quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide also includes a troubleshooting section and tear-out quantitative Western blotting checklist.

Let us show you just how easy getting good data can be. Fill out this form to be contacted by a life science expert today!

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