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Best practice when running SDS-PAGE

Choosing the appropriate gel:

  • Use a gel with a concentration that allows the proteins to move towards the middle or lower half of the gel. This will give a better visualization. As a rule, higher acrylamide concentrations are best-suited for lower molecular weights.
  • Use a single concentration gel for separating similar-sized proteins. This will allow a better visual, as the proteins will be more evenly spread throughout the gel.
  • Use a broad gradient gel when working on an unknown sample without a target protein or when separating proteins of different sizes. This will allow a broader range of proteins to be separated.

A table showing the appropriate acrylamide concentrations for gels with the purpose of separating proteins of particular sizes. To the left is a column for size ranges of proteins measured in kDa and to the right is a column for acrylamide concentration measured in percentage. The table shows that for separating proteins in the range of 4-40 kDa, the appropriate acrylamide concentration should be up to 20%. For proteins in the range of 12-45 dKa, a concentration of approximately 15%. For proteins in the range of 10-70 dKa, a concentration of 12.5%. For separating proteins of approximately 15-100 dKa, the appropriate acrylamide concentration should be around 10%. For proteins in the range of 50-200 dKa, a concentration of approximately 8% should be used and for proteins greater than 500 dKa, an acrylamide concentration of 4-6% is appropriate.

Figure 1. Acrylamide concentrations in gels: Protein size to acrylamide concentration in gels.

Preparing the sample:

  • Make the concentration of protein and loading buffer consistent between the samples to standardize the separation.

Loading the sample:

  • Place pipette in the bottom of the well without penetrating the gel and release the sample slowly to avoid overflow into the running buffer or neighboring wells.
  • Add more glycerol to the sample if the sample floats. This will make the sample more dense than the surrounding buffer, allowing it to fall to the bottom of the well.

Running the gel:

  • Choose a lower current to avoid running the gel too fast and overheating the gel.
  • Keep the current consistent throughout the run to standardize the separation.
  • Let the gel run until the dye front has migrated to the bottom of the gel, without running off the gel.

Staining the gel:

  • Use a large amount of fresh Coomassie koomarsi blue to stain the gel
  • Use a fresh batch of Coomassie koomarsi blue for each gel to ensure best staining