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SDS PAGE gel

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Harms Lab Wiki > Protein Expression & Purification > > SDS PAGE gel
Purpose: to assess protein purity
Protocol:
  1. In PCR tubes (strip tubes work very well), combine 15 μL protein with 15 μL gel loading dye for each sample you intend to run.  The gel loading dye is made by combining 95 μL Laemmli Sample Buffer and 5 μL β-mercaptoethanol (sometimes called 2-mercaptoethanol). 
  2. Heat the sample to 95 °C for 5 minutes on the thermocycler. (This, plus the SDS and β-mercaptoethanol, denature all of the proteins).  
  3. Load 20 μL of each sample into the appropriate lanes. 
  4. Load 10 μL of molecular weight marker (Thermo Scientific, Spectra Multicolor Broad Range Protein Ladder).  This should not be heated. 
  5. Run for 45 minutes at 200 V. 
  6. Break the gel out of its plastic case using the metal tool provided by Bio-Rad.  It is easiest to get the gel off by rinsing under DI H2O.
  7. Place to gel in a small plastic container (it works to use the top of a tip box) and cover in DI H2O.
  8. Microwave 1 min; place on shaker 1 min.
  9. Dump water out and repeat step 8 two more times (3 total rinses).
  10. Pour a small amount of SafeStain Coomassie gel stain over the top of the gel.  You don’t need a lot; just enough to fully cover the gel.
  11. Microwave for 1 min with a loose plastic lid.  (The lid prevents coomassie from coating the inside of the microwave). 
  12. Place on shaker for ~1 hr, then dump stain and cover with ddH2O to destain.
  13. Destain anywhere from 2 hr to overnight, and then image the gel.
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