Lecture Notes on Western Blotting and ELISA

Lecture Welcome and Announcements

  • Good morning and welcome back to QBM.
  • Discussion on Lectures 11 and 12 to finish up, with plans for Lecture 13 next week (not on the exam).
  • Exam details:
    • Open on Friday, due the following Friday.
    • No studies required during spring break following the exam.
  • Reminder: Allergy season is beginning; personal note on potential allergies to bayberries.

Western Blotting

MCAT Questions and Protein Kinase

  • Review of MCAT questions related to Western blotting, emphasizing details to note:
    • Protein kinase A, CREB, KREB327 discussed in context.
    • Phosphorylation details:
    • Phosphorylation occurs at serine 115 by glycogen synthase kinase III.
    • Note that serines can be phosphorylated.
  • Examination of wild-type versus the 119 mutant, highlighting results from Western blot
    • Wild-type phosphorylated by both proteins:
    • GSK3 + protein kinase A show bands in the blot.
    • 119 mutant shows no bands indicating no phosphorylation.

Understanding Western Blot Variants

  • Clarification on language:
    • Native gel vs. SDS-PAGE:
    • Native does NOT involve heat or beta-mercaptoethanol.
    • Reductive SDS-PAGE involves heat and beta-mercaptoethanol which breaks disulfide bonds.
  • Importance in evaluating denaturation of proteins and their interactions (disulfide bonds).

Direct vs. Indirect Western Blots

  • Preference for indirect Western blots:
    • Requires only one secondary antibody which amplifies the signal.
    • Different primary antibodies can be used with a single secondary.
  • Example of secondary antibodies: tagged with enzymes such as horseradish peroxidase (HRP).
    • HRP oxidizes substances using hydrogen peroxide, detecting with either tetramethylbenzidine (TMB) or luminol.
  • Discussion of alkaline phosphatase as another secondary option:
    • Use with substrate fibrom 4-chloro-3-indoyl phosphate, produces blue dimer upon phosphate removal; sensitivity noted.
  • Overview of detection methods:
    • Colorimetric (TMB, HRP), chemiluminescence (Luminol), and fluorescence (emerging technology with growing sensitivity).
  • Autoradiography:
    • Uses radioactive-labeled antibodies; high sensitivity but with handling precautions due to radioactivity.
  • Visualizing methods:
    • X-ray film for non-fluorescent images; darkroom process explained with backdrop of safety precautions.
    • X-ray film can be left on the blot for extended periods, efficient data collection noted.

Processing Steps in Western Blotting

  • Overview of full procedure:
    • SDS-PAGE for protein separation.
    • Blotting onto membrane, blocking with milk to prevent non-specific binding.
    • Addition of primary and secondary antibodies, enzyme-mediated visualizations.
  • SDS-PAGE markers' visibility issues on Western blots requiring dual gel runs for accuracy.
  • Special markers available for visibility in colorimetric and fluorescent detection.

Sensitivity and Quantification of Western Blots

  • The sensitivity range:
    • Western blotting detects proteins in the nanogram to femtogram range.
    • Exposure time affects detection levels (e.g., 5-second exposure detecting picograms vs. 5-minute detecting femtograms).
  • Transition from film to digital imaging for precision and data consistency discussed.
  • Summary of typical lab timing for SDS-PAGE (2-3 hours) and Western blot (4-5 hours), noting operational corner-cutting for efficiency.

Antibody Considerations

  • Primary antibodies target proteins of interest, whereas secondary antibodies target primary antibodies' Fc regions.
  • Overall procedure reflects on importance:
    • Establishing specificity and sensitivity of the antibody interactions.
  • Discussion on nonspecific aggregation and strategies to mitigate it through better washing protocols.

Applications and Diagnostic Use of Western Blotting

  • Critical use in cancer detection and infectious diseases, demonstrating the diverse applications of Western blotting in research.
  • Impact of methods on scientific literature and illustrations in textbooks, solidifying the role of Western blots in molecular biology research.

Conclusion and Laboratory Updates

  • Introduction of ELISA techniques following Western blotting to create diagnostic assays.
  • Historical context on radioimmunoassays leading to ELISA's non-radioactive version.
  • ELISA's rapid processing times compared to Western blots, exploring various ELISA types (direct, indirect, sandwich).

Future Learnings

  • Collaborations on ELISA and its curated use in diagnostic health settings discussed.
  • Closing remarks and encouragement about the upcoming lab sessions and final preparations for exams slated for the following week.
  • Next lecture scheduled to continue on Friday.