Making SDS-PAGE Gels

Last updated: 1/12/12 By: Tim Starr

Overview

You can either buy the Invitrogen NuPage prepoured gels, or pour your own gel using Invitrogen Gel Cassettes (1.0mm cat# NC2010). Or use any other electrophoresis apparatus. The gel we currently use is a Bis-Tris acrylamide gel. Generally I use a 10% acrylamide gel, but you can vary the percentage depending on the size of the protein you are looking for. The recipes below are adapted from Invitrogen.

Protocol

• Gather four gel cassettes in a rack, and four combs
• Prepare fresh Ammonium persulfate (APS) by mixing 100 mg in 2 ml H2O (5%).
• Mix the separating gel ingredients in a 50 ml conical. Add the TEMED and APS last, as the gel will immediately start to harden once these ingredients are added.
• Using a 10 ml pipette fill the gel casting 3/4 full (about 6 ml each). Do this fairly rapidly to avoid hardening of the gel
• Gently overlay the gel with H2O or isobutanol to ensure a smooth edge.
• Let gel harden for about 1 hr
• Pour off H2O/isobutanol and dry with kim wipes

Separating Gel Recipe for 4 gels (approximately 25 ml)

• Prepare the stacking gel in the same manner as the separating gel
• Rapidly fill the remaining 1/4 of the gel casting and insert the comb
• Wipe excess gel and let stacking gel harden for 1 hour.

Stacking Gel Recipe for 4 gels (approximately 12.5 ml)

• Wrap gel in wet paper towel and store in plastic bag at 4°C.