Chapter 4 Flashcards
What are the key steps in protein purification?
- Cell lysis (release proteins from cells).
- Centrifugation (separate components by density).
- Salting out (precipitate proteins at different salt concentrations).
- Dialysis (remove small molecules).
- Chromatography techniques (separate based on size, charge, or binding affinity).
How does a scientist release proteins from cells or tissues?
Homogenization (breaking open cells mechanically or chemically).
How does centrifugation help in protein purification?
Differential centrifugation separates cell components by density.
Ultracentrifugation measures protein mass, density, and shape.
How does salting out work?
Proteins precipitate at high salt concentrations, allowing separation based on solubility.
What is dialysis, and why is it used?
A semipermeable membrane removes small molecules (e.g., salts) while retaining proteins.
What are the three main types of chromatography used to purify proteins?
- Gel Filtration Chromatography – Separates proteins by size (larger proteins elute first).
- Ion-Exchange Chromatography – Separates proteins by charge.
- Cation-exchange: Negatively charged beads bind positively charged proteins.
- Anion-exchange: Positively charged beads bind negatively charged proteins. - Affinity Chromatography – Separates proteins based on specific ligand binding.
What is HPLC (High-Performance Liquid Chromatography)?
Uses finer beads and high pressure for faster, sharper separations.
What are the different types of electrophoresis used to separate proteins?
- Native PAGE – Maintains protein structure and charge.
- SDS-PAGE – Uses SDS detergent to denature proteins and separate by size only.
- Isoelectric Focusing – Separates proteins based on their isoelectric point (pI).
- Two-Dimensional Gel Electrophoresis –
- Step 1: Isoelectric focusing separates proteins by pI.
- Step 2: SDS-PAGE separates by size.
How is the specific activity of a protein fraction calculated?
Specific activity = Enzyme activity / Total protein concentration.
Should increase after each purification step.
What is the difference between polyclonal and monoclonal antibodies?
Polyclonal antibodies – Recognize multiple epitopes on a target protein.
Monoclonal antibodies – Recognize one specific epitope (produced from a single B-cell clone).
How are antibodies used in biochemical research?
Western Blot: Detects specific proteins after SDS-PAGE.
ELISA: Quantifies proteins using antibody-enzyme reactions.
Microscopy: Fluorescent-labeled antibodies track proteins in cells.
Co-immunoprecipitation: Identifies protein-protein interactions.
What is MALDI-TOF mass spectrometry, and how is it used?
MALDI (Matrix-Assisted Laser Desorption Ionization) ionizes proteins.
TOF (Time-of-Flight) measures mass-to-charge ratio (m/z) to determine protein identity.
What is Edman Degradation, and what is its limitation?
Stepwise degradation of peptides from the N-terminus.
Limited to ≤50 amino acids in sequence.
How are proteins sequenced when they are too long for Edman Degradation?
Break into smaller peptides using enzymes (e.g., trypsin).
Reassemble sequences using overlapping fragments.
What are the three main techniques used to determine protein structure?
- X-ray Crystallography – Uses X-ray diffraction to determine atomic structure.
- NMR (Nuclear Magnetic Resonance) – Studies proteins in solution (NOESY measures distances between atoms).
- Cryo-Electron Microscopy (Cryo-EM) – Uses frozen protein samples and electron beams to create 3D structures.
