Title: Learn: Pouring, Loading, and Running SDS-PAGE Gels
Course: BTEC-2020-301-302-303-F21 > Section: Protein Gels

Module2 Pouring, Loading, and Running SDS-PAGE Gels

Pouring an SDS-PAGE Gel

The polyacrylamide matrix is formed from a mix of acrylamide and bis-acrylamide.  These two chemicals are supplied together in one bottle (labelled 30% acrylamide/bis-acrylamide solution). You will mix this acrylamide/bis solution with Tris buffer (to provide Cl- ions and the appropriate pH), SDS (to keep the proteins coated with SDS), and some water (to bring everything to the right concentration and volume).  Finally, and only once you are ready to pour the gel, you will add ammonium persulfate (APS) and N,N,N',N'-tetramethylethane-1,2-diamine (TEMED). The addition of APS and TEMED causes the acrylamide and bis-acrylamide to polymerize into polyacrylamide; in other words, they cause the liquid solution to turn into a gel.

Liquid acrylamide is a neurotoxin, so it is important that you protect yourself by wearing gloves. Polymerized polyacrylamide (the gel form) is not a neurotoxin. However, since some non-polymerized acrylamide may still be present on a gel, you should only handle the gels with gloves on.

Loading and Running an SDS-PAGE Gel

It’s a bit trickier to load an SDS-PAGE gel than an agarose gel. In SDS-PAGE, the gel is sandwiched between two plates of glass, and the wells can be difficult to see. You will need to get your pipette tip between the glass plates within a well, and slowly dispense the sample.  Getting good at loading these gels takes practice, and it’s normal to make mistakes at first.  Some people like to use a yellow sample loading guide, which can help identify where the wells are; the loading guide can be seen in the video below.

Once your ladder and samples are loaded, set the power according to your protocol instructions, and begin the run. The gel is finished running when the dye front reaches the bottom of the gel.

SDS-PAGE gels are run, generally at ~180 volts, until the dye front from the loading dye hits the bottom of the gel. This generally takes 40 minutes to an hour. When the dye is at the bottom of the gel, power is turned off, and the gel is removed from the assembly. Most researchers cut off and dispose of the stacking gel, as it rips easily. The gel needs to be soaked in a stain, and then destained, to allow visualization of the proteins.

Watch: Video Demonstration

Pouring and Running SDS-PAGE (Jean Bower, 18:59)