Methods of Protein Analysis

Flashcards covering fundamental methods of protein analysis, including primary and tertiary structure analysis, and protein identification techniques discussed in the lecture notes.

Amino Acid Composition

A method involving acid hydrolysis and amino acid analysis to cleave peptide bonds, separate individual amino acid residues, and quantify their percentage in a protein or small peptide.

Edman Degradation

A method used for amino acid sequencing that provides N-terminal sequence information for approximately 15-50 residues, useful for protein identification and detecting post-translational modifications.

Proteolysis

The process of cleaving a protein into smaller peptides using chemicals or enzymes called proteases, which break the polypeptide backbone at specific amino acid residues.

Proteases

Enzymes that cleave polypeptide backbones at specific amino acid residues.

Trypsin

A commonly used protease that cleaves on the C-terminal side of basic residues (Arginine, Lysine).

Chymotrypsin

A commonly used protease that cleaves on the C-terminal side of aromatic residues (Tryptophan, Tyrosine, Phenylalanine).

Electrophoresis

A widely used method in biochemistry and molecular biology that separates macromolecules like peptides, proteins, DNA, and RNA based on their movement in an electric field.

Two-Dimensional Electrophoresis

A technique that achieves better resolution separation of complex mixtures by performing electrophoresis in one direction and then repeating it in a perpendicular direction, often with altered conditions.

Paper Electrophoresis

A method used to separate small molecules such as amino acids and peptides on paper, where their migration is determined by net charges influenced by the solution's pH.

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

A method that separates proteins based primarily on their mass. Sodium dodecyl sulfate (SDS) denatures proteins and imparts a negative charge proportional to their mass, allowing them to migrate through a polyacrylamide gel inversely proportional to the log of their molecular weight.

Isoelectric Focusing Gel Electrophoresis

A method used to separate proteins based on their isoelectric point (pI). Proteins migrate in a pH gradient until they reach a position where their net charge is zero.

Two-Dimensional Gel Electrophoresis (pI and MW)

A high-resolution method that combines isoelectric focusing and SDS-PAGE to separate complex protein mixtures first by their isoelectric point and then by their molecular weight.

Immunoblotting (Western blotting)

A method for the selective detection and/or quantitation of a specific protein in a complex mixture using antibodies. Proteins are separated by gel electrophoresis, transferred to a membrane, and then probed with specific primary and labeled secondary antibodies.

Mass Spectrometry

A powerful method for determining the precise mass of molecules. It is a core methodology in proteomics, used for identifying, quantifying proteins, elucidating chemical composition, amino acid sequence, and post-translational modifications.

Proteomics

The study of all proteins encoded by a genome.

X-Ray Crystallography

A method used to determine the three-dimensional structures of macromolecules at atomic resolution by analyzing the diffraction patterns produced when protein crystals scatter an X-ray beam.

Nuclear Magnetic Resonance (NMR) Spectroscopy

A method used to determine the structures of small proteins in solution by measuring chemical shifts and identifying positional relationships between atoms from isotopically labeled proteins in a magnetic field.