Haynes:PAGE

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SDS-PAGE

by Karmella Haynes, 2015

Principle: Proteins are denatured and given a negative charge with a detergent (SDS), disulfide bonds are broken with a redox agent, samples are loaded to the top of a vertical gel, then separated by protein fragment size by applying an electric charge.

MATERIALS

• Protein sample buffer (e.g. NuPAGE® LDS Sample Buffer 4X)
• Redox agent - 1M DL-Dithiothreitol (DTT) (Sigma D0632-1G)
• Polyacrylamide gel (use the appropriate gel for your application)
  • Bis-Tris - small to mid-size proteins
  • Tris-Acetate - large proteins
  • Tris-Glycine - "stacked" gel for running large and small proteins on the same gel
• Running buffer (use the appropriate buffer for your gel)
  • Bis-Tris gel - MES SDS or MOPS SDS (e.g., 20x NuPAGE® MOPS SDS Running Buffer, Life Technologies NP0001)
  • Tris-Acetate gel - Tris-Acetate SDS
  • Tris-Glycine gel - Tris-Glycine buffer (e.g., 20x Novex® Tris-Glycine SDS Running Buffer, Life Technologies)
• Antioxidant - Only if using NuPAGE buffer, NuPAGE® Antioxidant (Life Technologies NP0005)

EQUIPMENT

• Vertical gel electrophoresis chamber (e.g. Invitrogen XCell SureLock)
  • Note: make sure the gel cassette is compatible with the electrophoresis system

PROCEDURE

Prepare protein samples

1. The final volume of each loaded sample is typically 20 μL. Check the specifications of the gel for well capacity.
2. Add protein samples to 1.5 mL tubes. Dilute the stock proteins in an appropriate buffer. Make sure the protein sample volume = the final volume - (concentrated sample buffer volume + 1.0 μL 1M DTT)
   1. Example 1: 20 μL final loading volume - (5.0 μL 4x sample buffer + 1.0 μL 1M DTT) = 14.0 μL protein
   2. Example 1: 20 μL final loading volume - (10.0 μL 2x sample buffer + 1.0 μL 1M DTT) = 9.0 μL protein
3. Make a loading buffer master mix: multiplier = total number of lanes + 1 for pipetting error. Example: if using one gel with 12 lanes, the multiplier is 13
   1. Example - for a 12-lane gel, using 4x sample buffer: 5.0 μL 4x sample buffer * 13 = 65. 1.0 μL 1M DTT * 13 = 13. Add 65 μL 4x sample buffer + 13 μL 1M DTT into a 1.5 mL tube
4. Set up the protein samples: in fresh 0.5 mL tubes, add protein and sample buffer master mix.
   1. Example - for a 12-lane gel, using 4x sample buffer: Add 6.0 μL sample buffer master mix + 14.0 μL protein per sample.
5. If running fewer samples than the total number of lanes, make "dummy" samples: for instance, 6.0 μL of master mix + 14.0 μL water for each empty lane
6. Heat samples, excluding the protein standard(s), @ 100°C/ 5 min. Cool at room temp.

Prepare the gel and electrophoresis chamber

1. Prepare the running buffer: dilute the buffer to make 500 mL of 1x running buffer.
   1. Note: 500 mL is sufficient for a mini gel system. For a larger chamber, make 1 L of 1x buffer.
2. Remove strip from the bottom of the gel to expose it.
3. Secure the gel, exposed side facing the outer wall, inside the chamber. Note: If running a single gel, set up a dummy gel mold opposite the actual gel.
4. Use the 1x running buffer to fill the inner camber to the top (above the gel lanes). For a mini gel system, this will take about 200 mL.
5. IMPORTANT: If using NuPAGE buffer, add 500 μL Antioxidant into the buffer in the inner chamber drop-wise, evenly over the surface of the buffer.
6. Next, fill the outer chamber just so that the bottom of the gel is submerged.
7. Gently pull out the well comb and discard it.
8. Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips.
9. Run the gel @ 120 V until the dye front reaches the very bottom.

HELPFUL RESOURCES

• Gel setup: Video - Running the Gel on the XCell SureLock® system