Lecture 12 Study Notes: Protein Interactions and Techniques

Introduction

  • The speaker opens with a greeting and acknowledges the early morning time, congratulating students for attending.

  • Mentions the transition into daylight savings time and its timing in relation to spring break, expressing mixed feelings about the schedule.

  • Provides some context for the early wake-up call, relating it to summer class timings.

Class Schedule and Updates

  • Current date: March 9th

  • Reminder of Exam 2, scheduled for this Friday, with completion allowed throughout the week.

  • Students are urged to complete the exam before leaving for spring break to avoid complaints about missed deadlines.

  • Announcement of the upcoming DNA section to be introduced on Wednesday, which will not be included in the exam.

  • Notes that the course is more than halfway through and alerts students about the fast pace expected after spring break.

  • Invitation for questions before proceeding with Lecture 12.

Protein Interactions Overview

  • Introduction to protein interactions, emphasizing their necessity in understanding molecular networks.

  • Importance of studying interactions between proteins, DNA, and RNA.

Review of Previously Discussed Techniques

  • Size Exclusion Chromatography: Determines if a protein is a monomer or dimer based on size during elution.

  • Co-Immunoprecipitation (Co-IP): Utilizes antibodies to pull down proteins and their binding partners.

  • SDS-PAGE and Western Blots: Techniques for analyzing protein interactions.

  • Native Gels: Used for studying specific protein interactions, such as vinculin with alpha-actin.

Introduction of New Techniques

Pull-Down Assay

  • Defined as similar to Co-IP but utilizes tags instead of antibodies; essential for studying protein interactions.

  • Introduces bait (known protein) and prey (target proteins) terminology.

  • Example: Using GST (Glutathione S-Transferase) tagged proteins.

    • Process:

    • Attach bait protein to GST.

    • Wash with a cell lysate to allow binding to occur.

    • Use glutathione to elute bound interactions for further study (e.g., sequencing, mass spectrometry).

Tandem Affinity Assay

  • Further refinement of pull-down assay—utilizes multiple affinity tags for purifying interactions to a higher degree.

  • Process involves:

    • Initial binding to protein A on an IgG affinity column, followed by a wash to remove impurities.

    • A second binding to calmodulin column to refine samples further.

Far Western Blot

  • Similar to a Western blot but employs a primary binding partner instead of a primary antibody.

  • Requires a native gel as denatured proteins from SDS-PAGE would compromise results.

  • Process outlined:

    • Running protein of interest across a native gel.

    • Identifying whether it binds to the target based on retention in the gel.

    • Advantages: Can identify specific protein interactions effectively (e.g., breast cancer EGFR investigation).

Mass Spectrometry

  • Utilizes cross-linking to study interactions, enabling researchers to deduce molecular structures via resultant fragments.

  • Example: Cohesin protein interactions possibly leading to 3D structural understanding through the mass spectrometry analysis trails.

Yeast Two-Hybrid System

  • Designed to analyze existing and novel protein-protein interactions systematically.

  • Developed by a UCF professor; involves screening interactions with a reporter gene indicating successful coupling of bait and prey proteins.

  • Two domains of GAL4 are used to demonstrate reporter activation through interacting proteins, leading to observable color changes from yeast expression.

Microarray and Gene Chips

  • Used for high-throughput analysis of protein interactions and screening processes. Can analyze thousands of proteins simultaneously.

  • Mechanism involves proteins adhered to chips reacted with target compounds or drugs, which subsequently aids in evaluation through binding interactions.

Calorimetry

  • Key for understanding thermodynamic aspects of interactions between proteins.

    • Isothermal Titration Calorimetry (ITC): Measures heat changes occurring when proteins interact.

    • Differential Scanning Calorimetry: Increases temperature to derive melting points and structural dynamics.

  • The calculations from calorimetry data provide key insights into binding affinities and energies of interactions.

Surface Plasmon Resonance (SPR)

  • Another important method for studying binding interactions.

  • Works on the principle of observing light reflection changes due to protein binding on a membrane.

  • Generates kinetic binding data to evaluate interaction affinity and stability.

Protein-DNA Interactions

  • Numerous techniques available to analyze protein-DNA interactions, essential for transcription regulation and DNA repair processes.

Southwestern Blot

  • A combination of the Southern and Western techniques, enabling detection of protein-DNA interaction via protein blotting and DNA hybridization.

Electrophoretic Mobility Shift Assay (EMSA)

  • Technique to observe binding of proteins to nucleic acids by assessing migration shifts in gel electrophoresis.

DNA Footprinting

  • Method to determine exact protein binding sites on DNA by digestion analysis post protein incubation.

Chromatin Immunoprecipitation (ChIP)

  • Method for detecting protein-DNA interactions in a cellular context using cross-linking, lysis, and immunoprecipitation strategies.

Conclusion & Next Steps

  • Final thoughts highlight the importance of proper technique selection for various research contexts.

  • Reminder of scheduled class breaks and exams. Encourage preparation for upcoming lessons and materials, particularly for practical implementations.

  • Wishing students well, drawing the session to a close while prompting questions before dismissal.