Module_19

Definition: A technique that separates macromolecules based on their size and charge using polyacrylamide gels formed through chemical crosslinking of acrylamide and bisacrylamide.
Initiation: Polymerization is initiated by ammonium persulfate and the base TEMED.
Types of Gels:
- Vertical Slab Gels: Commonly formed structure for PAGE.
- Acrylamide Percentage Variability:
  - Lower Percentage Gels: Suitable for nucleic acids and isoelectric focusing (separation based on charge).
  - Higher Percentage Gels (Above 10%): Used primarily for protein separation by mass (SDS-PAGE).
  - Gradient Gels: Varying percentages used to enhance resolution in separations.

Techniques:
1. Staining:
  - Uses substances like coomassie blue or silver staining to visualize all proteins.
2. Autoradiography:
  - Incorporates radioactive labeling, e.g., using 35S-Methionine for detection.
3. Immunoblotting:
  - Utilizes enzyme-linked antibodies and substrates for sensitive protein detection (colorimetric or chemiluminescent).

Reducing Conditions: Involves agents like 2-mercaptoethanol to cleave disulfide bonds within and between proteins, improving protein linearization and accurate size estimation.
Comparison of Conditions:
- Reducing reveals distinct protein sizes, e.g., a protein with 50 kDa and 30 kDa subunits appears as a single 80 kDa band without reduction and as two distinct bands with reduction.
Effect of Crosslinking: Proteins chemically crosslinked can display as a single band on SDS-PAGE compared to distinct bands when unlinked.

Extraction Conditions: Proteins maintained in native state using mild detergents (e.g., Dodecyl-Maltoside).
Advantages: Preserves functional protein interactions as they exist naturally, allows recovery in active form.

Overview:
- Western blotting identifies specific proteins post separation via SDS-PAGE, followed by transfer to a solid support for detection.
- Highly sensitive and specific, utilizes selective antibody systems for detection based on molecular weights.
- Gel Structure: Comprises two layers: stacking gel (top) and separating gel (bottom) optimized for target protein size.
Quantification: Detection of protein changes over time through band intensity analysis in immunoblotting.

Purpose: Purifies specific antigens from complex protein mixtures using monoclonal or polyclonal antibodies.
Protocol: Forms immune complexes which are precipitated using beaded supports, followed by analysis via SDS-PAGE.

Co-immunoprecipitation: Requires controls for specific binding verification, including Input, Unbound, and IP samples.
Proximity-Dependent Labeling (BioID method): Detects transient protein interactions, employing biotin ligase fused to proteins of interest to label nearby proteins for purification and analysis.

Mechanism: Interaction detection between proteins by observing energy transfer from one fluorophore to another upon proximity.
Application: Visualizes dynamic interactions, e.g., calmodulin and myosin light chain kinase during calcium stimulation.

Principle: Laser-based technology enables cell counting, sorting, and analysis. Single cells are passed through lasers that detect scatter and fluorescence.
Flow Cytometer Setup: Involves sample preparation, staining with fluorescent antibodies, and calibration of optical settings.
Data Analysis: Captures data related to cell size and granularity, visualized through histograms and plots.

Health and Medicine:
1. Immunophenotyping: Identifying immune cell populations, crucial for diagnosing diseases like leukemia.
2. Monitoring Diseases: CD4+ T-cell counts in HIV patients guide therapy.
3. Cancer Applications: Tumor marker detection and circulating tumor cell analysis.
4. Stem Cell Research: Essential for regenerative medicine and differentiation analysis.
5. Drug Testing: Evaluates effects on cell behaviors during clinical trials.