NanoBio: Protein Gels

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(New page: ==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 ...)
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Revision as of 16:11, 8 July 2008

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.
    1. Each well holds ~50uL maximum. You can aim for 40uL load.
  2. Mix up enough dye with 1/10 BME for all of your samples.
    1. 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.
    1. Note: Tube clamps are suggested since the air might expand. Don't be alarmed if they pop on you!
    2. 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

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