A Simple Outline of Methods for Protein Isolation and Purification

Vocabulary flashcards covering core techniques, reagents, and principles of protein isolation, purification, and diagnostic analysis.

Phosphocellulose Purification

A chromatography purification method that utilizes positively charged residues to isolate DNA-binding proteins.

Chaotropic Reagents

Compounds such as urea and guanidine hydrochloride that enhance extraction efficiency by breaking down protein structure and dissolving readily in water, typically requiring high salt concentrations.

Density-Gradient Ultracentrifugation

A centrifugal separation method using media of differing densities to isolate specific cell types (such as immune cells, adipocytes, or keratinocytes), eliminate cellular impurities, or isolate cell organelles.

Salting Out

A simple protein isolation and stabilization technique that lowers protein solubility through competition for water using highly soluble salts such as ammonium sulfate, precipitating target proteins at specific salt concentrations.

Isoelectric Precipitation

A protein fractionation method in which the pH of a solution is adjusted using a mineral acid or trichloroacetic acid (TCA) until the target protein reaches its isoelectric point ($\text{pI}$) and precipitates.

Centrifugal Membrane Concentration

A single-step enrichment procedure for concentrating protein solutions that can experience membrane clogging and protein loss when processing highly concentrated samples.

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; an analytical technique that provides 1-dimensional separation of proteins according to their molecular size.

2-Dimensional Electrophoresis

A high-resolution proteomics method that combines isoelectric focusing (separating proteins by their $\text{pI}$ values) with subsequent SDS-PAGE (separating proteins by molecular size).

Western Blotting

An analytical method that detects specific target proteins separated by gel electrophoresis using target-specific antibodies.

Immunoprecipitation

An antibody-based enrichment technique where target proteins bind to specific antibodies captured by protein A- or protein G-conjugated agarose beads and are isolated through rounds of centrifugation.

ELISA

Enzyme-linked immunosorbent assay; a sensitive, antibody-mediated, enzyme-amplified detection method that identifies and quantifies trace antigens in bodily fluids without prior purification.

Nanobody (Single-Chain Antibody)

A camelid-derived antibody consisting of only two polypeptide chains whose antigen-binding site is formed by a single chain, facilitating simpler cloning and production compared to four-chain antibodies.

Aptamers

In vitro-synthesized RNA or DNA oligonucleotides selected to bind specifically to target proteins, functioning as alternatives to antibodies.

SELEX

Systematic evolution of ligands by exponential enrichment; an iterative screening process used to identify aptamers with high binding affinity and specificity for target molecules.

Mass Spectrometry Imaging

A technique (also called imaging mass spectrometry) that maps and detects proteins in situ directly within cells or tissues by generating 2-dimensional spatial mass profiles.