2D Gel Electrophoresis: A Step-by-Step Guide

Instructor Introduction

• Presenter: Steve Freebie, Staff Scientist at Bio-Rad Labs
• Purpose: Demonstrate how to run reproducible 2D gels.
• Video Segments:
  1. Applying sample and rehydrating the IPG strip.
  2. Equilibrating the IPG strips for the second dimension gel.
  3. Staining the second dimension gel.

Segment 1: Sample Preparation and Rehydration

• Tools and Materials:
  • Bio-Rad's 2D Starter Kit (includes E. coli sample and reagents).
  • Rehydration Buffer Components: Urea, CHAPS, DTT, and Bromophenol Blue.
• Procedure:
  1. Prepare Sample: Reconstitute 200 micrograms of E. coli sample with rehydration buffer.
  2. Rehydration of IPG Strip:
     • Load 200 microliters of sample into the focusing tray.
     • Use trays specific to the length of IPG strips.
     • Tilt the tray for even distribution of the sample.
  3. Load and Position IPG Strip:
     • Remove protective backing using forceps.
     • Position the strip with gel side down into the sample bead.
     • Ensure no air bubbles are trapped.
  4. Rehydration Methods:
     • Passive rehydration in focusing tray.
     • Active methods and cup loading for specific protein types.
  5. Use Protein IF Cell: Align electrodes, cover strips with mineral oil.
  6. Program the Cell: Typical rehydration: 12 hours; focusing: 6-8 hours.
  7. Post-Focusing: Store strips in rehydration tray at -80°C if not immediately proceeding to the second dimension.

Segment 2: Equilibration for Second Dimension

• Equilibration Buffers:
  • Buffer 1: Contains DTT for protein reduction.
  • Buffer 2: Contains iodoacetamide for protein alkylation.
  • Both buffers contain SDS for protein denaturation.
• Procedure:
  1. Blot Excess Oil: Remove oil from IPG strip with filter paper.
  2. Equilibrate Strips:
     • Use ReadyPrep 2D Starter Kit instructions.
     • Place strip in rehydration tray, add buffer 1, shake for 15 mins.
     • Repeat with buffer 2.

Segment 3: Running and Staining the Second Dimension Gel

• Gel Preparation:
  1. Pre-cast Criterion Gels: Suitable for 11 cm IPG strips.
  2. Prepare Gel:
     • Remove sealing tape and rinse wells with water.
     • Remove unpolymerized acrylamide.
  3. Load Strips:
     • Rinse IPG strip to remove equilibration buffers.
     • Position in gel well, ensure no air bubbles.
  4. Seal Gel:
     • Use Aurous overlay to seal strip and standard plug into well.
• Running the Gel:
  • Use Criterion Cell Box; load buffer, program for 200 volts, and run for 1 hour.
• Staining:
  1. Remove Gel: Open cassette, remove and prepare gel for staining.
  2. Stain with BioSafe Coomassie: Three 10-minute washes; stain for 45 mins to 1 hour.
  3. De-stain as Needed: Until desired background is achieved.

Conclusion

• Results: Clear background and well-resolved spots on gel.
• Tips: Clean samples and Bio-Rad’s 2D Starter Kit enhance reproducibility.
• Further Resources: Visit Bio-Rad's Expression Proteomics website for more tips and techniques.