Lecture Notes: Running Reproducible 2D Gels

Introduction

• Presenter: Steve Freebie, Staff Scientist, Bio-Rad Labs
• Objective: Demonstrate how to run reproducible 2D gels.
• Video Segments:
  1. Applying sample and rehydrating the IPG strip.
  2. Equilibrating the IPG strip for the second dimension gel.
  3. Staining of the second dimension gel.
• Goal: Gain tips, techniques, and tricks for quality 2D gels.

Segment 1: Sample Application and IPG Strip Rehydration

• Tools: Bio-Rad's 2D Starter Kit (includes reagents and E. coli sample).
• Rehydration Buffer Components: Urea, CHAPS, DTT, ampholytes, bromophenol blue.
• Sample Preparation:
  • Load 200 µg of E. coli sample in rehydration buffer onto an 11 cm pH 4-7 IPG strip.
• Rehydration Process:
  • Load sample into the focusing tray.
  • Use the right tray for the strip length (11 cm here).
  • Tilt tray for even distribution.
  • Remove plastic protective backing from IPG strip with forceps.
  • Place IPG strip into focusing tray gel side down.
  • Remove trapped air bubbles.
• Rehydration Techniques:
  • Passive rehydration in the focusing tray.
  • Active or cup loading for specific protein separations.

Segment 2: Equilibration of IPG Strip

• Procedure:
  • Remove IPG strip, blot excess oil.
  • Equilibration Buffers:
    • Buffer 1: Contains DTT for protein reduction.
    • Buffer 2: Contains iodoacetamide for protein alkylation.
  • Buffers also contain SDS for protein denaturation.
• Equilibration Steps:
  • Place IPG strip in rehydration tray, add buffers sequentially, shake for 15 minutes each.
• Preparing for Second Dimension:
  • Use Criterion pre-cast gels (11 cm strips).
  • Prepare gels by removing sealing tape, rinse wells, and remove unpolymerized acrylamide.

Segment 3: Running and Staining the Second Dimension Gel

• Running the Gel:

  • Position IPG strip in gel cassette.
  • Use Precision Plus Protein Standards for reference.
  • Seal with agarose overlay to prevent air bubbles.
  • Run in Criterion Gel Box at 200 volts for 1 hour.

• Staining Process:

  • Remove gel from cassette, perform initial rinses to remove SDS.
  • Stain with Bio-Rad's Bio-Safe Coomassie for 45-60 minutes.
  • Destain in water until desired background is achieved.

Conclusion

• Outcome: Generate clear background and well-resolved spots.
• Additional Resources: Visit Bio-Rad’s Expression Proteomics website for more tips and techniques on sample preparation and 2D gel electrophoresis.

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These notes summarize the key steps and insights from the demonstration on running 2D gels, highlighting the importance of each step to achieve good quality, reproducible results.