Antibodies, Protein Purification, and Protein Analysis - Vocabulary Flashcards

Vocabulary-style flashcards covering antibodies, purification techniques, and protein structure/analysis concepts from the provided notes.

B cells

Lymphocytes that develop in the bone marrow and differentiate into plasma cells that secrete antibodies.

Antibodies (immunoglobulins)

Y-shaped proteins with variable regions that bind specific antigens and can neutralize pathogens, trigger complement, or mark targets for immune cells.

Monoclonal antibodies

Identical antibodies produced from a single B cell clone that recognize only one epitope.

how are monoclonal bodies made?

by fusing antibody-producing B cells from an immunized animal with myeloma (tumor) cells

Polyclonal antibodies

A mixture of antibodies from multiple B cell clones that recognize multiple epitopes on the same antigen.

How are polyclonal antibodies obtained?

Injecting an animal with antigen and taking its blood every so often.

How are antibodies used in purification?

Antibodies can be attached to beads, resin, or a column to capture their specific target protein from a mixture.

Antibody purification via beads/columns

Antibodies attached to beads, resin, or a column to capture their target protein from a mixture.

Non-binding proteins during purification

Non-target proteins do not bind and are washed away.

What is immunoprecipitation?

technique where antibody–protein complexes are pulled down by beads and pelleted by centrifugation.

Affinity chromatography

Purification based on specific binding to a ligand immobilized on a column matrix.

Elution methods in affinity chromatography

Changing pH, ionic strength, or adding a competing ligand to release the target.

Western blot

Technique to detect presence, size, and relative abundance of specific proteins in a sample.

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis; separates proteins by size with SDS giving uniform negative charge.

Steps of a Western blot

SDS-PAGE → transfer to membrane → primary antibody → secondary antibody with enzyme → substrate for detection.

What determines amino acid properties?

R groups determine properties: nonpolar, polar, acidic, basic, or aromatic.

Peptide bonds

Covalent bonds formed by a dehydration reaction between the carboxyl group of one amino acid and the amino group of another.

Protein backbone vs. side chains

Backbone provides structure; side chains drive interactions and folding.

Protein structure levels

Primary: sequence; Secondary: α-helix and β-sheet; Tertiary: 3D folding; Quaternary: assembly of chains.

α-helix

Stabilized by H-bonds between carbonyl O and amide H four residues apart; side chains point outward. Does not use r groups

β-sheet

Stabilized by H-bonds between strands; can be parallel or antiparallel; side chains alternate above/below.Does not use r groups

Disulfide bonds

Covalent bonds between cysteine residues that stabilize protein structure, common in extracellular proteins.

Noncovalent interactions

Hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions that influence folding.

what holds a proteins shape?

Hydrophobic forces

Allosteric regulation

Regulatory molecule binds at a site other than the active site to alter enzyme activity.

Phosphorylation

Addition of a phosphate group that changes protein conformation and activity.

ATP hydrolysis in proteins

Powering conformational changes in motor proteins for movement.

Differential centrifugation

Repeating centrifugation steps to separate cellular components by size and density.

Velocity sedimentation

Separation by size/sedimentation rate using a gradient (e.g., sucrose).

Equilibrium sedimentation

Separation based on buoyant density, independent of particle shape.

Ion exchange chromatography

Separation by charge differences; elution by increasing salt or changing pH.

Gel filtration (size-exclusion) chromatography

Separation by size and shape; large molecules elute first, small molecules last.

SDS-PAGE details

SDS provides uniform negative charge; separates proteins by size.

Isoelectric focusing (IEF)

Separation by isoelectric point (pI); proteins stop migrating at their pI.

pI (isoelectric point)

The pH at which a protein has net zero charge.

2D-PAGE

Combination of IEF (by pI) and SDS-PAGE (by size) for high-resolution protein separation.

What are the main steps in a Western blot?

1.Separate proteins by SDS-PAGE. 2. Transfer to membrane. 3. Incubate with primary antibody. 4. Incubate with secondary antibody linked to enzyme. 5. Add substrate for detection.