Protein Purification Techniques: Ion Exchange, Reverse-Phase, and Affinity Chromatography

Vocabulary flashcards covering core concepts, terminology, resins, elution strategies, and ligands from lecture notes on ion exchange, reverse-phase, and affinity chromatography.

Ion exchange chromatography

A column chromatography technique used primarily to purify proteins by separating them based on their net electrical charges.

Isoelectric point ($pI$)

The specific pH at which a protein has no net charge and will not bind to charged column beads in ion exchange chromatography.

Cation-exchange chromatography

An ion exchange process in which the stationary phase consists of beads or resin with a fixed negative charge, used to bind positively charged molecules.

Anion-exchange chromatography

An ion exchange process in which the stationary phase consists of beads or resin with a fixed positive charge, used to bind negatively charged molecules.

Carboxymethyl (CM) cellulose

A common cation-exchange resin containing fixed negative charges used to bind positively charged species.

Diethylaminoethyl (DEAE) cellulose

A common anion-exchange resin containing fixed positive charges used to bind negatively charged species.

Gradient elution

An elution technique where salt concentration increases constantly and gradually during the elution process, such as moving from no $KCl$ to $1\,M$ $KCl$.

Step-wise elution

An elution technique where the change in salt concentration is abrupt without intermediate stages, such as switching directly from a no-salt loading buffer to a $1\,M$ elution buffer.

Myoglobin

In the carboxymethyl cellulose experiment, a protein that passes through the cation-exchange column without binding to the negatively charged beads.

Cytochrome c

In the carboxymethyl cellulose experiment, a protein that binds to the negatively charged beads and is eluted using step-wise elution with $1\,M$ $NaCl$.

Reverse-phase chromatography

A form of hydrophobic interaction chromatography where nonpolar molecules in an aqueous buffer bind to a hydrophobic matrix and are eluted by reducing buffer polarity.

C-18 column

A chromatographic column containing hydrophobic beads with $18\text{-carbon}$ chains, commonly used in reverse-phase chromatography.

Affinity chromatography

A highly effective column chromatography technique that separates molecules based on specific structural characteristics or biological activity using immobilized ligands on a support matrix.

Ligand

A specific biological molecule (such as an antibody, peptide, hormone, or substrate) immobilized on a support matrix in affinity chromatography to selectively bind target molecules.

$$2'5'\,ADP$$

An affinity chromatography ligand that specifically binds enzymes with $NADP^+$ as a cofactor.

Concanavalin A

An affinity chromatography ligand that specifically binds sugar residues on proteins.

Cibacron Blue (dye)

An affinity chromatography ligand that specifically binds enzymes with nucleotide cofactors.

Protein A (from bacteria)

An affinity chromatography ligand that specifically binds $IgG$ molecules.

Lysine

An affinity chromatography ligand that specifically binds ribosomal $RNA$.

Arginine

An affinity chromatography ligand that specifically binds some proteases.

Metal chelation

An affinity chromatography ligand interaction method that specifically binds histidine-tagged proteins.

Poly A or poly U

Affinity chromatography ligands that specifically bind nucleic acids.