Question 1

Which of the following best explains why 'salting out' is commonly used as an initial step in protein purification?

Accepted Answer

It selectively precipitates proteins by reducing their solubility at high ionic strength, allowing separation from other biomolecules.

Question 2

A protein has a pI of 5.0. If you want to purify it using anion exchange chromatography, which buffer pH should you choose?

Accepted Answer

$	ext{pH} = 7.0$

Question 3

Which of the following statements about SDS-PAGE is correct?

Accepted Answer

Proteins are separated based on their size, as SDS gives them a uniform charge-to-mass ratio.

Question 4

Why is ammonium sulfate commonly used for protein precipitation?

Accepted Answer

It is highly soluble and allows precise control of ionic strength for selective precipitation.

Question 5

In size-exclusion chromatography, which protein will elute first?

Accepted Answer

The largest protein in the mixture.

Question 6

A researcher adds imidazole to a Ni-NTA affinity column. What is the purpose of this step?

Accepted Answer

To compete with His-tagged proteins for binding to nickel, eluting the target protein.

Question 7

Which equation correctly describes electrophoretic mobility ($\mu$) in gel electrophoresis?

Accepted Answer

$\mu = \dfrac{q}{f}$

Question 8

A protein sample is prepared for SDS-PAGE by adding SDS and $\beta$-mercaptoethanol, then heating. What is the main effect of this treatment?

Accepted Answer

It gives all proteins a similar shape and charge-to-mass ratio, allowing separation by size.

Question 9

Which detection method is most sensitive for visualizing proteins after electrophoresis?

Accepted Answer

Silver Stain

Question 10

You run a size-exclusion chromatography (SEC) and observe one peak, but SDS-PAGE of the peak shows two bands. What is the most likely explanation?

Accepted Answer

The proteins form a complex that is separated by size in SEC but dissociate in SDS-PAGE.

Question 11

Which of the following best describes the principle of affinity chromatography?

Accepted Answer

Proteins are separated based on their specific binding to a ligand attached to the stationary phase.

Question 12

A protein with a pI of 9.0 is placed in a buffer at pH 7.0. What is the net charge of the protein, and which ion exchange resin would bind it?

Accepted Answer

Net positive; binds to cation exchange resin.

Question 13

Which of the following is a key advantage of using size-exclusion chromatography for protein purification?

Accepted Answer

It preserves the quaternary structure of proteins.

Question 14

During protein purification, why is it important to minimize denaturation and degradation?

Accepted Answer

To maintain the protein's native structure and function for downstream applications.

Question 15

Which of the following combinations would best separate a mixture of proteins differing in both size and charge?

Accepted Answer

Ion exchange chromatography followed by size-exclusion chromatography

Question 16

A protein is purified using anion exchange chromatography at pH 8.0. Which property of the protein is most critical for its binding to the column?

Accepted Answer

The protein must have a net negative charge at pH 8.0.

Question 17

Which of the following best describes the effect of increasing salt concentration during elution in ion exchange chromatography?

Accepted Answer

It disrupts ionic interactions, causing bound proteins to elute.

Question 18

Which of the following is NOT a factor that must be considered to maintain protein stability during purification?

Accepted Answer

Protein's codon usage

Question 19

A mixture contains four proteins: Albertase (5,000 Da, pI = 9.5), Halifaxatin (35,500 Da, pI = 6.0), Lethbridge Phosphatase (7,000 Da, pI = 9.0), and Ontario Kinase (103,000 Da, pI = 5.5). Which protein would elute first from a size-exclusion column?

Accepted Answer

Ontario Kinase

Techniques of Protein Purification: Principles and Methods

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