SDS-PAGE. You know the acronym. You run gels every day. You know what to do, but do you know why you do it?
You load your sample, start the current, watch for the bubbles, see the blue dye front move down the gel. Have you ever wondered what is happening?
We have the answers to your SDS-PAGE questions. This blog addresses some of the biggest ones and gives a basic explanation.
Some of the basics:
SDS is a detergent that unfolds proteins and coats them in negative charge.
Why negative charge?
The negatively charged proteins will want to move towards the positively charged anode, but the speed at which they do so is based on size, not charge. The coating of SDS molecules gives every protein the same net negative charge.
Why are there two gel layers?
They have different functions. The stacking layer (where the samples are loaded) has a lower pH and lower acrylamide content. Its main function is to get all of the proteins lined up and condensed so they enter the gel at the same time. The resolving layer (the big one where the bands appear) has a higher pH and a higher acrylamide content. Its main job is to separate the proteins by their molecular weight.
Why do the layers have different pHs?
To control ionic state. The pH of the gel layer affects the ionic state (charge) of ions in the buffer. When the electrodes are turned on, the ions start moving towards the anode. The speed at which they move is dependent on their charge state. It is important that they move at different speeds in each of the gel layers.
Why do the gel layers have different acrylamide percentages?
To control protein movement. The acrylamide percentages of the layers affect how fast the unfolded proteins move through the gel. The lower percentage of the stacking layer allows them to move more freely and line up together before entering the resolving layer. The higher percentage in the resolving layer slows them down and forces them to separate based on molecular weight.
Why are there so many bubbles?
H2 and O2. Once the electric current is applied, the anode and cathode are involved in redox reactions that remove electrons from water molecules in the running buffer, resulting in gas formation.
Those are the basics! Read all of the specifics in my full-length article.