CH. 5 -Protein Purification

Why must proteins be released from cells before purification?

To access the protein of interest and remove contaminants.

How does a blender help in protein isolation?

Breaks open cells and organelles in buffer.

What does a homogenizer do?

Breaks cells by squeezing homogenate through a tight plunger.

What is sonication used for?

Sound waves break open cells.

How does freeze-thaw break cells?

Cycles of freezing and thawing rupture membranes.

Why are detergents sometimes needed in cell lysis?

To detach membrane-bound proteins.

What is differential centrifugation?

Separation by spinning at increasing speeds to pellet components.

What is ammonium sulfate precipitation (“salting out”)?

Proteins lose their solvation shell and precipitate due to reduced hydration.

Why does increasing ammonium sulfate separate different proteins?

Different proteins precipitate at different salt concentrations.

What is column chromatography?

Separation based on different interactions with stationary and mobile phases.

What is the stationary phase in chromatography?

Solid material packed in the column.

What is the mobile phase?

Solution that carries sample through the column.

What causes slower movement in chromatography?

Stronger interaction with the stationary phase.

What does gel filtration chromatography separate by?

Size.

Which molecules elute first in gel filtration?

Large molecules (excluded from pores).

Which molecules elute last in gel filtration?

Small molecules (enter pores).

What is V₀ in size-exclusion chromatography?

Void volume for molecules excluded from beads.

What is Vₑ?

Elution volume for molecules entering the beads.

What is affinity chromatography based on?

Specific binding between a protein and a ligand.

What happens to non-target proteins in affinity chromatography?

They flow through without binding.

How is the target protein eluted in affinity chromatography?

With soluble ligand or by changing pH/ionic strength.

What does ion-exchange chromatography separate by?

Net Charge.

What binds to cation-exchange resins?

Positively charged proteins.

What binds to anion-exchange resins?

Negatively charged proteins.

How are bound proteins eluted in ion exchange?

Changing pH or adding salt (Na⁺ competes).

What is percent recovery?

Amount of protein retained in each purification step.

What is specific activity?

Enzyme activity per mg protein; indicator of purity.

What is HPLC used for?

Fast, high-resolution purification.

What is reverse-phase HPLC?

Nonpolar stationary phase with polar mobile phase.

What indicates a good purification step?

A high increase in specific activity.

What is fold purification?

Specific activity of a step divided by the previous step.

Why is affinity chromatography often the best step?

It gives the largest purification in one step.

What is electrophoresis used for?

Separation by charge-to-size ratio.

What gel is used for proteins?

Polyacrylamide.

What gel is used for nucleic acids?

Agarose.

How do molecules migrate in electrophoresis?

Negatively charged molecules move toward the positive electrode.

What does SDS do in SDS-PAGE?

Denatures proteins and coats them with uniform negative charge.

In SDS-PAGE, what determines movement?

Size — small proteins migrate faster.

What is isoelectric focusing?

Separation of proteins by isoelectric point (pI).

When does a protein stop moving in isoelectric focusing?

At its pI, where net charge = 0.

What is primary structure determination?

Identifying amino acid composition and peptide sequence.

What does trypsin cleave?

After Lys and Arg (basic residues).

What does chymotrypsin cleave?

After aromatic residues (Phe, Trp, Tyr).

What does cyanogen bromide cleave?

At methionine residues.

How are protein sequences determined from fragments?

Using overlapping peptide fragments.

What is Edman degradation?

Sequencing method that removes the N-terminal residue stepwise.

What is ELISA used for?

Protein detection using antibodies.

What is a Western blot?

Proteins separated by SDS-PAGE and transferred to a membrane for detection.

What are protein chips?

Microarrays with thousands of immobilized proteins.

What is proteomics?

Study of all proteins and their interactions.

What is structural proteomics?

Examining protein structures.

What is expression proteomics?

Comparing protein expression across conditions.

What is interaction proteomics?

Studying how proteins interact with other molecules.