Protein properties and purification

What physical properties of proteins can be used to distinguish them

Protein size, shape, isoelectric point (pI), surface charge, hydrophobicity, polarity, and ligand-binding properties.

Why is a pure protein preparation required

Other proteins and molecules in a mixture can interfere with measurements of a protein’s properties or activity, so purification allows the protein to be studied more reliably.

What is protein purification

The process of separating a protein of interest from other cellular components and proteins.

What is a crude extract

A mixture obtained after cells or tissues are disrupted, containing proteins and other cellular components.

What is tissue or cell disruption

The mechanical or chemical breaking open of cells to release their contents into an aqueous buffer.

What are lysozyme and DNase I used for in bacterial cell disruption

They help break down bacterial cell components and DNA, respectively, during preparation of a cell extract.

What is centrifugation

A separation method that uses centrifugal force to separate components according to their sedimentation behaviour.

What is the principle of centrifugation

Heavier or larger particles generally sediment faster than lighter components under the same conditions.

What is a pellet

The material that sediments to the bottom of a centrifuge tube, often containing cellular debris.

What is a supernatant

The liquid remaining above the pellet after centrifugation.

What is a cleared lysate

The supernatant obtained after centrifuging a cell lysate to remove much of the cellular debris.

What is controlled protein precipitation

A purification step that separates proteins based on differences in their solubility.

What is salting out

A technique that uses high salt concentrations to decrease the solubility of some proteins, causing them to precipitate.

What is an ammonium sulfate cut

A precipitation step in which ammonium sulfate is added to a chosen saturation level to precipitate certain proteins selectively.

Why can ammonium sulfate separate different proteins

Different proteins have different solubilities at different salt concentrations, so they precipitate under different conditions.

What is dialysis

A technique that removes small dissolved molecules, such as salts, from a protein solution through a selectively permeable membrane.

What principle allows dialysis to work

Small molecules can pass through the membrane, while larger molecules such as proteins are retained, depending on the membrane’s molecular-weight cutoff.

What are the initial steps in protein purification

Disrupt cells or tissue, centrifuge the extract, selectively precipitate proteins if appropriate, and remove excess salt by dialysis when needed.