⚡ Core Concept: Analytical Tools for Cellular Proteomics and Genomics
Molecular cytology utilizes high-precision biochemical assays to isolate, quantify, and visualize macromolecular dynamics within cellular compartments. Core workflows include Western Blotting (immunoblotting), Flow Cytometry / FACS, Enzyme-Linked Immunosorbent Assay (ELISA), and Immunofluorescence (IF).
1. Western Blotting Workflow (Immunoblotting)
Cells lysed with RIPA buffer containing protease/phosphatase inhibitors; proteins linearized and negatively charged using SDS and heated with β-mercaptoethanol (cleaves disulfide bonds).
Proteins resolved across a polyacrylamide gel matrix exclusively based on molecular weight (mass-to-charge ratio equalized by SDS).
Electric field transfers separated protein bands from polyacrylamide gel onto a solid PVDF or nitrocellulose membrane support.
Membrane blocked with 5% BSA/non-fat dry milk to prevent non-specific binding; probed with primary antibody followed by HRP-conjugated secondary antibody for chemiluminescence (ECL).
Figure 1: Western Blot Gel Transfer & Chemiluminescent Detection
Protein Analysis
Western blot setup: electrophoretically resolved proteins immobilized on PVDF are identified by primary antibodies and secondary antibodies coupled to horseradish peroxidase (HRP).
2. Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS)
Flow cytometry enables multi-parametric quantitative interrogation of thousands of individual cells per second in suspension:
- Hydrodynamic Focusing: A sheath fluid aligns single cells into a narrow, single-file laminar stream passing a laser interrogation point.
- Forward Scatter (FSC): Light scattered at small angles proportional to cell size / cross-sectional area.
- Side Scatter (SSC): Light scattered at 90° angles reflecting internal granularity and membrane complexity.
- Fluorescence Channels (FL): Fluorophore-conjugated antibodies (FITC, PE, APC) quantify cell surface markers (CD3, CD4, CD8) or intracellular DNA content (propidium iodide for cell cycle phase analysis).
🎓 High-Yield Exam Checkpoint
- Blotting Summary Mnemonic (“SNOW DROP”):
- Southern → DNA (Edwin Southern)
- Northern → RNA
- O → O (No match)
- Western → Protein
- FACS Gating in Immunology: CD4+ Helper T cells vs CD8+ Cytotoxic T cells are identified by dual-fluorophore staining gating on lymphocytes.
