Detecting cyt c using TMBZ (heme) staining of PAGE gels

Author: Heather Jensen

Goal: Determine molecular weights of Cytochromes c from whole cells

Materials

  • LDS 4x protein loading dye
  • Protein ladder
  • Samples
  • Protein gel; typcially 4-20% PAGE
  • 6.3 mM TMBZ in methanol, made fresh! (TMBZ is stored at 4° C)
  • 0.25M sodium acetate, pH 5.0. (can be made from a 1M stock)
  • 30% hydrogen peroxide (9.79M, stored at 4 °C)
  • isopropanol

Critical Steps:

  • Select a percentage gel that will allow you to clearly identify your cytochrome c of interest. There is a chart in 5204 near the gel running boxes that shows the different gel types and the resolution provided by each. Choose an appropriate gel so you can see the proteins you want.
  • Modify the SDS-PAGE protocol to permit staining of cytochromes c. Specifically, it is essential to avoid addition of reducing agents, which will reduce the disulfide bond holding the heme c to the polypeptide backbone. Additionallywe have found that hemes are better stained when LDS is used instead of SDS, when the gel is run at lower voltage/current.

Procedure:

Preparing Samples

  1. Do protein expression, then isolate the cellular fraction of interest (whole cell, periplasm, or membrane. See

  2. DO NOT

    • Add BME or other reducing agents — this will reduce the heme, removing the peroxidase activity

  3. Add 4x LDS dye when gel is ready to load. Should stand no longer than 15 minutes.

    • 10uL dye per 30uL sample

  4. Load Samples onto Gel

  5. Run the PAGE

  • in the incubator or a water bath at 16°C

  • ________% PAGE

  • 200V for 60-70 minutes. (until pink dye from LDS runs off the gel)

TMBZ staining of gel

  1. Solutions you need for the stain:

    • 6.3mM TMBZ in methanol: ______g TMBZ in 30mL methanol.

    • 0.25M sodium acetate, pH 5.0

    • 30% hydrogen peroxide (9.79M, in the fridge)

    • Isopropanol

  2. Make the TMBZ solution while the gel is running. This takes time to solubilize. Cover the flask in foil and leave on a stir plate, stirring while the gel runs.

  3. Rinse the gel with water for 5 minutes

  4. Immediately before use, mix 30mL TMBZ solution with 70mL sodium acetate solution (per gel)

  5. Immerse the gel in the solution above. put gel in the dark. (ie a drawer)

  6. Mix occasionally (every 15 minutes) for 1-2 hours

  7. Add 495 μL 30% hydrogen peroxide (final concentration 30 mM in 100 mL). Mix well.

  8. Staining should be visible within 3 minutes and increases in intensity over the next 30 minutes.

  9. Background of the gel may be removed by destaining with 3:7 isopropanol:0.25M sodium acetate. This mixture can be replaced once or twice with fresh solution. Gels may also be stored in this solution.

Detection of Bands:

  1. Place gel in plastic sheet protector.

  2. Place gel in scanner.

Note: This gel can be stained with imperial afterwards to get a total protein stain. 

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