W19 L2 - Protein Purification Techniques

Flashcards reviewing key concepts related to protein purification techniques discussed in the lecture, including size exclusion chromatography and ion exchange chromatography.

What is size exclusion chromatography (SEC)?

A method that separates proteins based on their size using porous plastic beads in a column.

In size exclusion chromatography, which proteins elute faster—large or small?

Large proteins, because they cannot enter the pores in the beads.

How can a standard curve be generated to determine the size of an unknown protein using size exclusion chromatography?

Plotting the log of molecular weight against elution time.

What is a key advantage of size exclusion chromatography regarding protein activity?

The protein is not denatured and remains functional.

What is ion exchange chromatography?

A method that separates proteins based on their net charge at a given pH.

How does ion exchange chromatography work to separate proteins?

Proteins with opposite charges bind to the matrix, while proteins with the same charge flow through.

How are proteins eluted from an ion exchange column?

By adding salt, which competes with the protein for binding to the charged matrix.

What is the difference between anion exchange and cation exchange chromatography?

Anion exchange chromatography involves a positively charged matrix that binds negatively charged proteins (anions). Cation exchange chromatography uses a negatively charged matrix to bind positively charged proteins (cations).

How can protein concentration be monitored as it elutes from a chromatography column?

Using a UV lamp and detector to measure absorbance at 280 nm, indicating the presence of aromatic amino acids and thus protein.

Why is SDS-PAGE often used in conjunction with ion exchange chromatography?

To check the purity and composition of the fractions collected during chromatography.