How SDS-PAGE Separates Proteins

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Have you ever wondered how SDS-PAGE separates proteins? SDS-PAGE is the first critical first step in the Western blotting process. It is an acronym for sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The net charge, molecular radius, and magnitude of an applied electric field determine the movement of any charged species through the field. In their native state, different proteins with the same molecular weight migrate at different speeds depending on their charge and molecular makeup.

Difference between SDS-PAGE and Western blotting

SDS-PAGE separates proteins by their molecular weight using a gel matrix, with help from an electrical field. Western blotting is a term used to describe the process of detecting a particular protein.

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 protein travels through the gel

The protein ladder is purchased to ensure it undergoes quality control and is properly prepared and ready for use. Experimental samples, like your proteins, must be prepared in the lab. Your goal when preparing your samples for Western blotting is denaturation and uniform coating the proteins with a negative charge.

Let’s go over the general steps of SDS-PAGE (Figure 1). To begin, first you will pipette your protein samples into the gel. Then, using a gel running system, like the Azure Aqua (shown below), an electrical currant will separate the proteins. SDS will provide denaturing condition and coat the negatively charged proteins, so that they will now move throughout the gel based on their size. The last step of SDS-PAGE is for you to detect and analyze the proteins based on their migration patterns to the molecular weight proteins.

How to separate proteins with SDS-Page. SDS-Page separates proteins from complicated samples of mixture. The detergent SDS and a heating step determine that the electrophoretic mobility of a single kind of protein is only affected by its molecular weight in the porous acrylamide gel
To separate proteins from complex mixtures, SDS-PAGE can be used. SDS-PAGE utilizes the detergent SDS and a heating step to ensure that the electrophoretic mobility of a particular protein is only influenced by its molecular weight within the porous acrylamide gel. (Created with BioRender.com)

These steps allow the proteins to travel effectively through the gel, based on their molecular size and weight toward the positive anode. Note that the SDS-treated proteins will move different rates due to their differences in molecular weight. The higher molecular weight proteins move slower through the porous acrylamide gel than the lower molecular weight proteins do.

How electrophoresis separates proteins

During the electrophoresis step, the proteins travel through a gel matrix, inside a gel running system (like an Azure Aqua). An electric current inside of the Aqua pushes proteins through the gel. The current shoves the proteins to equilibrium, where they are  unable to move.

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

Common issues that occur 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. 

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

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
quantitative western blot basics

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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.

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