<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Getting Started

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You have samples and you want to see (1) what proteins are in that sample and (2) how much of each protein there is. You can use this protocol to semi-quantitate protein sizes and concentrations in your samples. You can use this information to roughly measure protein purity.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Protocol

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• [ ] Denature samples.
  • [ ] Label one tube per sample.
  • [ ] Add 4 uL of 6x Laemmli sample buffer and 20 uL of sample (~1 ug total protein) per tube. Mix by pipetting.
  • [ ] Incubate samples at 90C / 10 min.
• [ ] Set up a gel box.
  • [ ] Open an individually wrapped protein gel (we use 10% - 20% Tris Gly gels).
  • [ ] Remove plastic tape at the bottom of gel. This exposes a strip of the gel to the running buffer, allowing current to flow from one electrode through the gel to the other electrode.
  • [ ] Place gel in gel box and seal tightly. Check the seal by pouring running buffer (we use Tris Gly) into the front half of the reservoir and checking that no buffer leaks into the back half. If so, remove the gel, empty the reservoir and try again.
  • [ ] Pour running buffer into the back half of the reservoir.
  • [ ] Carefully, remove comb from top of gel.
• [ ] Load 10 uL protein ladder onto the outermost lanes.
• [ ] Load each sample onto its own lane. Target between 1-2 ug purified protein and between 10 uL and 20 uL per lane.
• [ ] Run the gel at 200 V / 40-50 min, or until the dye front touches the bottom of the gel.
• [ ] Stain and destain the gel.
  • [ ] Using a gel knife, crack open the plastic cassette holding the gel.
  • [ ] Remove the gel carefully into a waterproof container. We find this is easier if you cover the bottom of your container with Ultrapure water (≥ 3 mm).
  • [ ] Cover the gel with Ultrapure water (~40 mL), microwave for 30 seconds, and rock for 5 minutes. Decant and repeat this step twice, washing a total of three (3) times.
  • [ ] Cover gel in a rapid protein stain (~40 mL) in the same container. We use PageBlue Protein Staining Solution. Microwave for 30 seconds, then incubate at room temperature with rocking for ~30 minutes.
  • [ ] Decant staining solution, rinse gel at least once, then cover in Ultrapure water. Incubate gel at room temperature with rocking until you can see distinct protein bands (up to overnight).
    • Notes: Use Kimwipes to speed up destaining
• [ ] Observe gel. High purity protein preps should have distinct bands at the correct molecular weight, with no unexpected additional bands.
  • Notes: you can use a gel imager to semi-quantitate your protein

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Resources and References

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<aside> <img src="/icons/megaphone_gray.svg" alt="/icons/megaphone_gray.svg" width="40px" /> Credits

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