Separate proteins on an SDS-PAGE gel

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

Overview

This protocol uses the Invitrogen XCell II Blot. Read that manual for more in-depth information. Can use several different types of buffers depending upon the gel.

Protocol

Prepare electrophoresis chamber

• Use a sharpie to make outlines of the wells on the gel cassette
• Remove the comb from the gel
• Remove the white tape on the bottom of the gel
• Assemble the electrophoresis chamber with one or two gels
• Fill the inner chamber with the appropriate buffer. If using NuPage prepoured or our own BIS-Tris gels use MOPS based buffer (Invitrogen Cat # NP0001) by mixing 40 ml of 20x buffer into 760 ml H2O. Otherwise use the Running Buffer below
• Make sure there is no liquid leaking into the outer chamber

Running Buffer (SDS-PAGE Acrylamide/Bis gels) 1x 1 L

• Note: Can use SDS at 0.1%
• Fill outer chamber with running buffer. If you're using NuPage buffer fill the chamber full. If you're using the homemade buffer just need to fill past the holes on the gel cassette. If you're using NuPage buffer add 500 µl of NuPage anti-oxidant to inner chamber.
• Wash out the wells by gently pipetting up and down in each well.

Prepare Protein lysates

• Calculate amount of protein to use. Add water to protein samples to bring total volume to 24 µl.
• Prepare Laemmli buffer by adding either 30 µg Dithiothreitol (DTT) or 20 µl Beta-mercaptoethanol (BME) as a reducing agent to 500 µl of Laemmli. Note, can store Laemmli 5x buffer at -20ºC, but add DTT or BME just before use.
• Add 6 µl of Laemmli buffer to each protein sample

Laemmli 5x buffer 1 ml (based on Maniatis A8.42 Cell lysis 1x SDS buffer)

• Load gel with ladder and protein samples (total volume 30 µl).
• Connect power supply to electrophoresis chamber and turn on power. This can be done in a 4ºC walk-in cooler. Voltage depends on the buffer and how fast you want to run the gel. Generally start with 80 V for 10 minutes to get the protein through the stacking gel and into the separating gel. After that can run at 100 to 125 Volts (which is usually around 7 to 8 mAmps). Normally takes about 3 hours for protein to run through gel (blue dye line at bottom). If using NuPage gels can set voltage 200 V.