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.
- 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)
- Vertical gel electrophoresis chamber (e.g. Invitrogen XCell SureLock)
- Note: make sure the gel cassette is compatible with the electrophoresis system
Prepare protein samples
- The final volume of each loaded sample is typically 20 μL. Check the specifications of the gel for well capacity.
- 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)
- Example 1: 20 μL final loading volume - (5.0 μL 4x sample buffer + 1.0 μL 1M DTT) = 14.0 μL protein
- Example 1: 20 μL final loading volume - (10.0 μL 2x sample buffer + 1.0 μL 1M DTT) = 9.0 μL protein
- 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
- 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
- Set up the protein samples: in fresh 0.5 mL tubes, add protein and sample buffer master mix.
- Example - for a 12-lane gel, using 4x sample buffer: Add 6.0 μL sample buffer master mix + 14.0 μL protein per sample.
- 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
- Heat samples, excluding the protein standard(s), @ 100°C/ 5 min. Cool at room temp.
Prepare the gel and electrophoresis chamber
- Prepare the running buffer: dilute the buffer to make 500 mL of 1x running buffer.
- Note: 500 mL is sufficient for a mini gel system. For a larger chamber, make 1 L of 1x buffer.
- Remove strip from the bottom of the gel to expose it.
- 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.
- 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.
- 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.
- Next, fill the outer chamber just so that the bottom of the gel is submerged.
- Gently pull out the well comb and discard it.
- Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips.
- Run the gel @ 120 V until the dye front reaches the very bottom.
- Gel setup: Video - Running the Gel on the XCell SureLock® system