W19 L1 - Protein Analysis and Purification

Flashcards reviewing protein analysis using gels, denaturation methods, and protein purification techniques discussed in the lecture.

Why is protein analysis more complicated than DNA analysis when using gels?

Proteins lack a uniform charge and shape, unlike DNA, which is uniformly negatively charged with a consistent structure.

What methods can be used to denature proteins?

Extreme pH, organic solvents (acetone, ethanol), detergents (SDS), and heat (boiling).

What is SDS (sodium dodecyl sulfate) and how does it aid in protein gel electrophoresis?

SDS is a negatively charged detergent that binds to proteins, unfolding them and providing a uniform negative charge, allowing separation by size.

Why is beta-mercaptoethanol (beta-ME) used in protein sample preparation for gel electrophoresis?

Beta-ME is a reducing agent used to break disulfide bonds in proteins, which cannot be disrupted by boiling alone.

What does PAGE stand for and what is it used for?

PAGE stands for Polyacrylamide Gel Electrophoresis and is a technique used to separate proteins based on their size.

How can a protein complex with a known molecular mass be analyzed using SDS-PAGE with and without beta-mercaptoethanol?

By comparing the resulting band patterns, it's possible to determine the subunit composition, whether subunits are linked by disulfide bonds, or whether the links are made with weaker non-covalent bonds.

What is a kilodalton (kDa)?

Kilodalton (kDa)is a unit of molecular mass equal to 1000 Daltons, often used to describe the size of proteins. A Dalton is one gram per mole.

What is the purpose of a marker lane in protein gel electrophoresis?

A marker lane contains proteins of known sizes, used as a standard to estimate the molecular weights of unknown proteins in the sample.

How do you create a standard curve to determine the size of unknown proteins from gel electrophoresis data?

Plot the log of the molecular weight of marker proteins against their migration distance, and use the resulting equation to estimate the size of unknown proteins.

What is the Hofmeister series, and what is its relevance to protein purification?

The Hofmeister series ranks ions based on their ability to salt in (increase solubility) or salt out (decrease solubility and precipitate) proteins.

What is salting out, and which salt is commonly used to precipitate proteins?

Salting out is the process of precipitating proteins by adding high concentrations of salt (typically ammonium sulfate) to reduce their solubility.

Why is ammonium sulfate used for protein precipitation?

Ammonium sulfate effectively precipitates proteins without denaturing them, allowing for recovery of active proteins.