NanoBio: Protein Gels

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Contents

Protein Gels

Materials

  • Protein loading dye (we have 4X)
  • Beta mercaptoethanol (BME, a reducing agent)
  • Protein ladder
  • Samples
  • Protein gel

Selecting the right gel

  • There is a chart in the room with 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.

Preparing your samples

  1. Normalize the protein concentrations. So if you know Sample A is a more dilute than Sample B, you can mix some B with water to normalize it to the lower concentration sample.
    • Each well holds ~60uL MAX. You should aim for 40uL load.
  2. Mix up enough dye with 1/10 BME for all of your samples.
    • Example: Your dye is 4X. You'll need 40/4 = 10uL for each sample. So mix up 9uL dye + 1uL BME.
  3. Add Dye+BME mix to your samples.
  4. Heat samples to 95 C for 5 minutes.
    • Note: Tube clamps are suggested since the air might expand. Don't be alarmed if they pop on you!
    • This process breaks disulfide bonds and more or less linearizes the proteins so shape doesn't affect running.
  5. Pulse centrifuge to collect condensation.

Load and run

  1. Open up the gel box and stick your gel in. It holds up to two gels.
  2. Add running buffer to the 2 cavities at the ends.
  3. Load samples. If you are not using all the wells, you may want to add loading buffer and water (~30uL total volume) to bordering lanes. This will reduce gel "smiling".
    • Ensure that you load ladder and samples in a fashion so you can track top/bottom and left/right!
  4. Run at 200V for 1hr.

Staining (using Imperial stain)

  1. Add water to cover the gel and shake on an orbital shaker for 5 minutes.
  2. Shake the stain bottle
  3. Cover the gel with stain.
  • Option 1: Microwave (quick but not as dark) 6-12ng sensitivity
    1. Place gel into a microwave-safe container and microwave for up to a minute.
      • Don't boil it. If it boils, just let it sit at just preboil temperature for the remainder of the time.
    2. Pour out dye (you can save it and reuse it if you store it in a dark/Al-foil'd container)
    3. Add water and microwave for a minute.
  • Option 2: Overnight (tried and true) up to 3ng sensitivity
    1. Staining and washing are done with the orbital shaker. Use diH2O for washing.
Resolution Stain Wash
<3ng 2 hours overnight
3-6ng 1 hour 1-2 hours
6-12ng 5-10 min 15 minutes

Source: PSR Protein Gels

Arthur K Yu 20:44, 8 July 2008 (UTC)

Heme Stain

Quick points for heme staining of gels

  • Use LDS over SDS
  • Run gel at a colder temperature
  • DO NOT use reducing agents

Sample Prep

  1. Do protein expression/extraction (either by B-PER or periplasmic isolation)
  2. DO NOT
    • Add BME -- this will reduce the heme, removing the peroxidase activity
    • Heat samples (?)
  3. Add 4x LDS dye when gel is ready to load. Should stand no longer than 15 minutes.
    • 10uL dye per 30uL sample

TMBZ (peroxidase) Stain directly of a PAGE gel

  1. Run the PAGE
    • in the incubator at 16°C
    • ________% PAGE
    • 200V for 60-70 minutes
  2. 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
  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 495uL 30% hydrogen peroxide (final concentration 30mM in 100mL). 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.

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