Amino Acids, Peptides, and Proteins - Techniques and Analysis

Vocabulary terms and definitions regarding protein purification, analysis, sequencing, and structural determination methods as covered in the lecture notes.

Gel filtration chromatography

A technique that separates proteins based on size, also known as size exclusion chromatography, where large proteins emerge from the column before small proteins.

Anion exchange chromatography

A method used to purify negatively-charged proteins by using positively charged beads.

Cation exchange chromatography

A method used to purify positively-charged proteins by using negatively charged beads.

Affinity Chromatography

A separation method based on a protein's ability to bind to a specific natural small molecule termed a ligand.

Unit of activity

A measurement defined as the number of $\mu\text{mol}$ of substrate converted to product per minute at $25\,^{\circ}\text{C}$.

Specific activity

The ratio of enzymatic activity to the total amount of protein, which increases as the protein becomes purer.

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, a technique where a detergent unfolds proteins and gives them a uniform negative charge so they separate based only on size.

$\beta$-mercaptoethanol (BME)

A reagent used to break disulfide bridges in proteins, often utilized in reducing SDS-PAGE.

Isoelectric focusing (IEF) PAGE

Electrophoresis used to experimentally determine a protein's isoelectric point (pI) by distributing proteins along a pH gradient based on their charge.

Protein UV Absorbance

A quantification method where aromatic amino acids, specifically tryptophan and tyrosine, absorb light near a maximum of $280\,\text{nm}$.

Beer's Law

The mathematical relationship $A = \epsilon \cdot c \cdot l$ used to determine protein concentration via spectrophotometry.

Bradford Assay

A protein quantification assay using a Coomassie-based reagent with an assay range of $125\text{--}1,500\,\mu\text{g/mL}$.

BCA Assay

A protein quantification assay that utilizes a copper conversion mechanism with an assay range of $20\text{--}2,000\,\mu\text{g/mL}$.

Western Blotting

A technique where proteins transferred from an SDS-PAGE gel to a membrane are identified using primary and secondary antibodies.

ELISA

Enzyme-linked Immunosorbent Assay, a technique using antibody-enzyme complexes to detect specific antigens, available in direct, indirect, sandwich, and competitive formats.

Edman Degradation

A classical protein sequencing method involving successive rounds of N-terminal modification and cleavage, which is limited to peptides of approximately $50$ amino acids.

Mass Spectrometry (MS)

A modern sequencing method using ESI MS or MALDI MS to precisely identify the mass and amino acid sequence of a peptide, including post-translational modifications.

Trypsin

A protease that fragments polypeptide chains by cleaving at the carbonyl (C) side of Lysine ($Lys$) and Arginine ($Arg$) residues.

Cyanogen bromide

A chemical reagent used for fragmenting polypeptide chains by cleaving at the carbonyl (C) side of Methionine ($Met$).

Homologs

Related protein sequences that include paralogs (found in the same species) and orthologs (found in different species).

X-ray Crystallography

A method for determining 3D protein structure by collecting diffraction data from a crystal to calculate electron density; it has no size limits but cannot see hydrogens.

NMR Spectroscopy

A structural determination method carried out in solution that can see hydrogens and capture protein movement, but works best with small, soluble proteins.

Cryo-EM

Cryo-electron microscopy, a technique for determining high-resolution structures of large complexes and membrane proteins in solution without the need for crystallization.