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SDS Page Gel with Coomassie Stain

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to run protein samples on an SDS Page Gel and visualize the proteins with a Coomassie Blue Stain.

Reagents and Materials

  • NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gels
  • 20X MES SDS Buffer
  • 4X LDS Sample Buffer
  • Protein Sample
  • Protein Ladder
  • Coomassie Brilliant Blue R-250 Staining Solution
  • Coomassie Brilliant Blue R-250 Destaining Solution
  • DI-H20
  • 1.5mL Eppendorf Tubes

Things to Note before starting

Waste

Any liquids collected during the wash with Coomassie Blue should be saved and submitted to EH&S for safe disposal.

Estimated Timing

While sample prep and an running the gel might only take a ~30 minutes. Staining and de-staining the Coomassie Blue Stain will take up to 24 hours.

Procedure

Sample Prep

  1. Prepare a 1X MES SDS Buffer solution by mixing 50mL of the 20X MES SDS Buffer with 950mL of DI-Water.
  2. In a 1.5mL Eppendorf Tube, combine 15uL Protein Sample with 5uL of 4X LDS Sample Buffer.
    1. If you want to add a reducing agent, use 13uL Protein Sample, 2uL Reducing Agent, and 5uL of the 4X LDS Sample Buffer.
  3. Mix samples by pipetting up and down.
  4. Incubate samples at 90C for 5 minutes.

Preparing the Gel

  1. Set up the SDS Page Gel Box.
  2. Pour 1X MES SDS Buffer to the fill line of the Gel Box, making sure the sample input wells are below the fill line.
  3. Add each sample to the appropriate well.
  4. Add a protein ladder to the wells on either end.
  5. Place the lid on the box, it should only fit in one orientation. .

Running the Gel

  1. Set the voltage 200V and run for ~40 minutes.
  2. Bubbles should immediately form, indicating that the current is running.
  3. You should be able to see the Protein Ladder and the dye from the Sample Loading Buffer as an indication of how far the proteins have run. When this has run through 80% of the gel, stop the electrophoresis.

Staining the Dye

  1. Remove the Gel from the SDS Page Box and place in a plastic container.
  2. Using a screwdriver or metal weigh spoon, pry apart the plastic surrounding the gel.
  3. After all the joints have been pried open, the top layer should be able to be removed.
  4. Using a razer, cut the bottom part of the Gel where the protein has not gone into.
  5. Carefully peel off the gel from the other side of the plastic directly into a plastic reservoir filled with DI-H20. ==DO NOT TEAR THE GEL! ==.
  6. Empty the reservoir with the Gel, being careful not to toss the gel.
  7. Wash the gel two more times with DI-H20 Washes.
  8. Add Coomassie Blue Stain to the plastic reservoir so that it covers the gel.
  9. Incubate on a tabletop rocker for 2 hours.
  10. Remove Coomassie Blue Stain and replace with Coomassie Blue Destainer.
  11. Incubate on a tabletop rocker for 1 hour.
  12. Repeat the process with more Coomassie Blue Destainer 4 times. With the last time, let it incubate on the tabletop rocker overnight.
  13. The gel should be mostly clear and you should be able to see blue marks in the gel where the Coomassie Blue has interacted with the protein in the gel.
  14. Take the gel to the visualizer in the Hutchinson Building to get an image of the gel.

Next Steps

The gel can be saved so long as it is submerged in either de-staining solution or water. However, it should be tossed if all analysis is complete.

Appendix

Footnotes