BCH 3303 - Chapter 6: Protein Purification and Characterization

Vocabulary practice flashcards covering protein hierarchy, purification techniques (solubility, size, charge, binding affinity), antibody assays (ELISA, Western blotting), and primary structure determination methods (Edman degradation, MALDI-TOF) from BCH 3303 Lecture 5.

Levels of Protein Structure

The organizational hierarchy of proteins comprising primary (amino acid sequence), secondary (regular backbone hydrogen-bonding patterns), tertiary (complete 3D spatial folding), and quaternary (multi-subunit spatial arrangement) structures.

Proteome

The functional representation of the genome, consisting of the entire set of expressed proteins (exceeding $1{,}000{,}000$ proteins) in a given cell type or biological condition.

Lactate Dehydrogenase Assay

An enzymatic assay that monitors protein activity by measuring the production of NADH, which uniquely absorbs light at a wavelength of $340\,nm$, during the conversion of lactate to pyruvate.

Specific Activity

The ratio of enzyme activity to the total amount of protein present in a mixture, which increases as a protein sample becomes progressively purified.

Differential Centrifugation

A cell fractionation process using sequential high-speed centrifugal force spins (ranging from $500 \times g$ to $100{,}000 \times g$) to separate subcellular fractions into crude pellets and cytosol based on size and mass.

Salting In

The biological phenomenon where most proteins require a low concentration of dissolved salt in solution to effectively dissolve in water.

Salting Out

A protein fractionation technique in which increasing salt concentrations alter water-protein interactions, causing different proteins to selectively precipitate out of solution based on solubility.

Dialysis

A protein purification method utilizing a semipermeable membrane submerged in an excess buffer solution to remove surplus small salt ions while retaining larger proteins.

Gel-Filtration Chromatography

A separation technique (also known as molecular exclusion chromatography) that isolates proteins by size using porous beads; small molecules enter internal bead spaces and elute slowly, whereas large molecules bypass the beads and elute quickly.

Ion Exchange Chromatography

A chromatographic method that separates proteins based on their net charge using charged matrix beads, causing oppositely charged proteins to bind while similarly charged proteins flow through.

Affinity Chromatography

A highly specific purification technique exploiting selective binding interactions between a target protein and a chemical group attached to matrix beads (e.g., glucose-binding proteins binding to matrix glucose residues).

High Pressure Liquid Chromatography (HPLC)

An enhanced column chromatography technique applying high pressures to finely divided matrices to obtain superior chromatographic resolution and rapid separation.

Electrophoresis

The migration of charged biomolecules through a gel matrix subjected to an electric field, influenced by electric field strength, net protein charge, and buffer pH.

SDS-PAGE

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; a denaturing gel technique that imparts a uniform negative charge to unfolded proteins, allowing them to separate strictly according to mass.

Isoelectric Point ($pI$)

The specific pH value at which a protein carries a net electric charge of zero.

Isoelectric Focusing

An electrophoretic technique separating proteins along a pH gradient established in a gel matrix until each protein migrates to its distinct isoelectric point ($pI$).

Two-Dimensional Gel Electrophoresis

A high-resolution protein separation method combining relies on isoelectric focusing in the first dimension (separation by $pI$) and SDS-PAGE in the second dimension (separation by molecular weight).

Ultracentrifugation

An advanced centrifugation process operating at very high speeds (up to $300{,}000 \times g$) that facilitates the isolation of small cellular organelles, vesicles, and macromolecular complexes.

Antigen

Any substance capable of stimulating an immune response and inducing the production of specific antibodies.

Epitope

The specific chemical structure or region on an antigen that is directly recognized and bound by an antibody.

Polyclonal Antibodies

A heterogeneous collection of antibodies derived from multiple B-cell lines that recognize multiple different epitopes present on a single antigen.

Monoclonal Antibodies

A homogeneous population of identical antibodies produced by a single clone of B cells that specifically target a single distinct epitope.

Indirect ELISA

An Enzyme-Linked Immunosorbent Assay method designed to detect and quantify the presence of specific antibodies in a biological sample.

Sandwich ELISA

An ELISA technique using immobilized monoclonal antibodies to trap a specific antigen, which is subsequently detected using an enzyme-linked secondary antibody.

Western Blotting

An analytical method in which gel-separated proteins are transferred onto a polymer membrane and probed with labeled antibodies to identify specific target proteins.

Edman Degradation

A chemical method for protein sequence determination that selectively labels and cleaves the N-terminal amino acid using phenyl isothiocyanate without breaking other peptide bonds.

MALDI-TOF Mass Spectrometry

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry; an analytical technique that measures mass-to-charge ratios ($m/z$) of laser-ionized proteins to accurately determine molecular mass.