Untitled Notes

High Performance Liquid Chromatography (HPLC)
- Components involved:
- Solvent: Communicates with the column.
- Pump: Drives the mobile phase through the system.
- HPLC Column: The space where separation occurs.
- Injector: Introduces samples into the mobile phase.
- PC for Data Acquisition: Captures and processes data from detection.
- Detector: Monitors the compounds as they elute.
- Waste: Manages leftover materials.
- Process Overview:
1. Sample is introduced into column with mobile phase.
2. Separation occurs by different moving speeds of each compound.
3. Detector connected to the outlet monitors eluting compounds and generates a chromatogram.

Western blotting
- Identifies protein antigens after SDS-PAGE.
ELISA
- Detects and quantifies proteins in a sample.
Immunofluorescence microscopy
- Localizes specific proteins within cells.
Immunoprecipitation
- Isolates specific proteins along with their binding partners.

Definition: Antibodies are proteins produced by immune system cells (B lymphocytes) that target foreign substances known as antigens—example: protein coat of a virus.
Components of an Antibody:
- Antigen
- Epitope
- Antigen-binding Site
- Heavy Chain
- Light Chain
- Variable Regions: Allow for specificity against diverse antigens.
- Constant Regions: Provide structural stability.

Concept: Selecting antibodies as bait to isolate targeted proteins from a whole proteome.
- Utilizing:
- Immunoprecipitation (IP) with motif antibodies for mass spectrometry analysis.
- Data analysis for understanding protein interactions.

Procedure:
1. Wells are pre-coated with a capture antibody.
2. Sample added for antigen binding.
3. Primary antibody binds to immobilized antigen.
4. Enzyme-labeled antibody attaches to the Fc region of the detection antibody.
5. Colorimetric reaction achieved from the substrate catalyzed by the enzyme, producing a colored product.

Process Steps:
1. Attach antibody to beads via protein A or G.
2. Lyse cells to release antigens and their binding partners.
3. Mix cell lysate with antibody-coated beads (antibody binds to antigen).
4. Purify by centrifugation to collect antigen and binding partners.

Types:
- Native PAGE: Preserves the natural state of proteins; inconsistency in migration through gel due to retained associations.
- SDS-PAGE: Imparts a negative charge proportional to molecular weight; denatures proteins and disrupts cross-links (using reducing agents such as DTT or BME).

Steps for Measurement:
1. Run molecular weight markers alongside an unknown protein on the gel.
2. Create a graph comparing molecular weight versus distance migrated.
3. Analyze the distance migrated by the unknown to estimate its molecular weight using the graph.

Allows verification of protein expression, relative amounts in samples, and analysis of protein-protein interactions.

Components of Fluorescence Microscopy:
- Excitation and emission filters, eyepiece, dichroic mirror, objective lens.
Fluorophores: Absorb light energy causing electrons to reach an excited state and release energy as light upon returning to the ground state.

Fluorescence Resonance Energy Transfer (FRET)
Fluorescence Recovery After Photobleaching (FRAP)
Multiphoton Excitation Microscopy: Engages two or more photons for fluorescent dye excitation; used in living cells.
Super-resolution Microscopy (e.g., STORM): Breaks diffraction limits to achieve resolutions down to 10-100 nm.

Peptide Fragmentation and Identification:
- Utilizes mass spectrometry to fragment proteins and analyze amino acid order using software.
Protein Labeling: A method for identifying and quantifying differential protein abundance; utilizes mass spectrometry techniques.

Material Flows in Cells:
- DNA: Potential of a cell.
- RNA: Current direction of activity in the cell.
- Proteins: Functional capabilities.
- Metabolites: Limiting currency for cellular processes.

Access online tools for protein exploration by protein name, 3D visualization available at: https://www.rcsb.org/