lab 13 western blot 11/26

Overview of Lab Experiments and Procedures

  • The lab focuses on protein analysis techniques, specifically SDS PAGE and Western blotting.
    • Students are expected to document their experimental results comprehensively.
    • Two specific tasks involve capturing images of gels and analyzing protein concentration.

SDS PAGE Gel Preparation

  • Students should take a picture of the SDS PAGE gel before leaving the lab.
  • Importance of the SDS PAGE Gel:
    • Acts as a visual representation of separated proteins based on size.

Protein Concentration Measurement

  • The lab report requires:
    • A growth curve.
    • A table of absorbances over time to evaluate protein concentration.
  • Includes details from the protein purification steps:
    • Utilizing nickel for purification.
    • Bradford method for protein concentration calculation (standard and individual concentration).

Western Blot Technique

Basic Concept

  • The Western blot technique involves multiple steps for detecting proteins.
  • It is named in reference to a similar technique developed by Southern for DNA (Southern blotting) and another for RNA (Northern blotting).
    • Western Blot specifically targets proteins.
  • No equivalent "Eastern" method is available as the central dogma of molecular biology primarily addresses DNA, RNA, and proteins.

Steps to Perform Western Blot

  1. Transfer Proteins from Gel to Nitrocellulose Membrane
    • This is the initial step where proteins are moved from the gel to a solid membrane for further analysis.
    • Indications of a successful transfer:
      • If proteins remain visible on the SDS PAGE gel, it suggests incomplete transfer.
      • Observing a ladder (marker) may indicate that not all proteins have transferred fully.
  2. Blocking Membrane
    • The purpose of blocking is to prevent non-specific binding during subsequent antibody incubations.
    • Blocking Agent:
      • Typically uses a 5% solution of dried non-fat milk (available from stores).
      • The proteins in the milk coat the membrane, reducing background noise during detection.
  3. Incubation with Primary Antibody
    • The primary antibody is specific to the target protein (e.g., GFP).
    • Process of generating antibodies:
      • Inject pure target protein into a host animal (e.g., mouse) to stimulate antibody production.
      • After 6-8 weeks, collect and purify the serum containing these antibodies.
    • Structure of Antibodies:
      • Y-shaped molecules with heavy and light chains that bind to specific antigens.
  4. Washing Step
    • Wash out unbound primary antibodies using the blocking solution to proceed with clarity for the next incubation.
  5. Incubation with Secondary Antibody
    • The secondary antibody is:
      • Specific to the primary antibody's host.
      • Conjugated with a reporter enzyme (e.g., alkaline phosphatase, AP) that helps visualize the target protein.
    • Functionality of Alkaline Phosphatase:
      • Converts yellow substrate into a purple color, indicating the presence of the target protein on the membrane.
    • Visualization process:
      • Introduction of a substrate that reacts with alkaline phosphatase to produce a visible color change.
  6. Quantification of Protein Levels
    • Western blots can show protein expression levels, with band brightness correlating to quantity.
    • Common practice includes comparing to loading controls like actin for normalizing data.
    • Calculation of protein levels might be presented as a ratio (e.g., p53 over actin).

Research Implications and Techniques

  • In a research context, Western blots are used to assess changes in protein expression under various conditions (e.g., drug treatment).
  • Different detection methods based on sensitivity include:
    • Use of horseradish peroxidase (HRP) for chemiluminescence, requiring advanced imaging technology.
    • Traditional approaches visible via direct staining for specific proteins.
    • Quantification of protein amounts using specialized equipment for high sensitivity analysis.

Upcoming Lab Tasks and Reporting

  • Students are required to:
    • Submit lab reports by the end of the week following the experiments.
    • Prepare for a quiz on Western blotting procedures next week.
    • Allocate a 20-minute timeframe for the quiz.
  • Instruction to take photos of gels as part of the documentation process to validate results.