Haynes:PAGE: Difference between revisions
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'''Prepare the gel and electrophoresis chamber''' | '''Prepare the gel and electrophoresis chamber''' | ||
# Prepare the running buffer: dilute the buffer to make 500 mL of 1x buffer. | # Prepare the running buffer: dilute the buffer to make 500 mL of 1x running buffer. | ||
# Remove strip from the bottom of the gel to expose it. | # 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. | # 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. | ||
# '''IMPORTANT''': If using NuPage buffer, measure about 200 mL 1x running buffer into a graduated cylinder or beaker, add 500 μL Antioxidant, swirl to mix. Use this to fill the inner chamber in the next step. | |||
# Fill the inner camber to the top (above the lanes) and the outer chamber just so that the bottom of the gel is submerged. | # Fill the inner camber to the top (above the lanes) and the outer chamber just so that the bottom of the gel is submerged. | ||
# Gently pull out the well comb and discard it. | # Gently pull out the well comb and discard it. | ||
# Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips. | # 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. | # Run the gel @ 120 V until the dye front reaches the very bottom. | ||
</div> | </div> |
Revision as of 18:41, 19 March 2015
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
- 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
- Antioxidant - Only if using NuPAGE buffer, NuPAGE® Antioxidant (Life Technologies NP0005)
EQUIPMENT
- Vertical gel electrophoresis chamber (e.g. Mini protean Bio-Rad)
PROCEDURE
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
- Calculate the number of lanes used = protein samples + protein standard(s). If this is less than the total number of lanes, prepare "dummy" samples (sample buffer + DTT + water) to fill empty wells
- 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.
- 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.
- IMPORTANT: If using NuPage buffer, measure about 200 mL 1x running buffer into a graduated cylinder or beaker, add 500 μL Antioxidant, swirl to mix. Use this to fill the inner chamber in the next step.
- Fill the inner camber to the top (above the lanes) and 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.