Lab Techniques in Molecular Biology

Lab Session 10: Expression of Fusion Protein from Positive Clones, SDS-PAGE, and Western Blot: Part II

  • Goal: Stain the membrane with Ponceau Red to confirm protein transfer to nitrocellulose and complete the western blot started previously.
I. Introduction
  • Antibodies: Multifaceted tools for protein study in various assays (e.g. western blot, immunofluorescence, ELISA).
    • Produced by the immune system, typically raised in rabbits (polyclonal) or mice (monoclonal).
    • Specificity varies; antibodies designed for similar regions of different proteins may cross-react.
  • Study Focus: Visualize GST::EGFP fusion protein among cellular proteins from bacterial clones.
  • Procedure Overview:
    • Previous week involved SDS-PAGE to separate proteins.
    • Today’s experiment: Perform a western blot to visualize the target protein.
Western Blotting Process
  1. Transfer proteins to nitrocellulose membrane.
  2. Block membrane to prevent non-specific binding (using casein or BSA).
  3. Primary antibody: Added, binding specifically to protein of interest.
  4. Unbound antibody washed off.
  5. Secondary antibody added (HRP-conjugated) to allow visualization.
  6. Visualization methods:
    • Enhanced Chemiluminescence (ECL): Luminol oxidized by HRP, creating luminescent signal.
    • Colorimetric Detection: Uses chloronaphthol to yield purple bands where antibody binds (indicative of the GST::EGFP fusion protein at approx. 59 kDa).
II. Laboratory Exercises
A. SDS-PAGE and Western Blot, Part II
  • Ponceau Stain: Visualize total protein pattern on the membrane.
    1. Stain membrane with Ponceau Red, then rinse with dH2O.
    2. Observations to note: band positions, loading uniformity, air bubbles.
  • Blocking Step: Critical for reducing non-specific antibody binding.
    1. Wash and then incubate in a blocking solution.

Lab Session 11: Extraction of Recombinant Protein from Escherichia coli and Purification Using a Glutathione Affinity Column

  • Goal: Lyse bacterial cells (IPTG-induced) to express GST::EGFP, then purify using affinity chromatography.
I. Introduction
  • Importance of cell lysis: to access intracellular proteins.
  • Methods:
    • Physical: Sonication, freeze-thawing, French press.
    • Chemical: Treat with reagents (e.g., B-PER with lysozyme).
  • After lysis, target protein purifying via affinity chromatography.
Affinity Chromatography Overview
  1. Sample Injection: Mixture of proteins including GST::EGFP.
  2. Adsorption: GST::EGFP binds to glutathione-linked resin.
  3. Wash: Remove non-specifically bound proteins.
  4. Elution: Target protein dislodged with reduced glutathione.
II. Laboratory Exercises
  • Growing Bacteria: Initial culture in medium followed by IPTG induction.
  • Harvesting Cultures: Centrifuge tubes to pellet bacteria and discard supernatant.

Lab Session 12: Analysis of Purification Fractions

  • Goal: Analyze purification fractions using SDS-PAGE and quantify GST::EGFP.
I. Introduction
  • Analysis Techniques:
    • SDS-PAGE to evaluate purity and presence of fusion protein in elutions.
    • Quantification: Fluorescence measurements or Bradford Assay.
A. Protein Quantification by Fluorescence
  • Measure fluorescence of GST::EGFP, examining excitation (488 nm) and emission (507 nm) wavelengths using a microplate reader.
  • Create standard curve using known concentrations of EGFP to compare with unknowns.
B. Protein Quantification by Bradford Assay
  • Measures total protein in sample using Coomassie dye.
  • Understand its sensitivity and compatibility with samples, particularly how detergents may impact results.
  • Use standard curves for quantification, avoiding BCA due to interference from glutathione.
II. Laboratory Exercises
A. SDS-PAGE of Purified Fusion Protein
  • Use staining method or stain-free gels for protein visualization.
Summary of Key Points
  • Understand the process of protein transfer and visualization in western blotting, the importance of purification techniques, and the methods for quantifying protein concentrations effectively.