Question 1

Which of the following best explains why the purification of proteins from natural sources, such as the pancreas for insulin, is often inefficient and requires large amounts of starting material?

Accepted Answer

The protein of interest is usually present at low concentrations relative to total protein, and each purification step results in some loss of material.

Question 2

A researcher is using ammonium sulfate to precipitate proteins from a cell extract. What is the principle behind the 'salting out' technique?

Accepted Answer

High salt concentrations compete with proteins for water, reducing protein solubility and causing precipitation.

Question 3

During dialysis, a protein solution is placed in a dialysis bag with a 10 kDa molecular weight cut-off and immersed in buffer. Which of the following will occur?

Accepted Answer

Small solutes and salts will diffuse out of the bag, while proteins larger than 10 kDa remain inside.

Question 4

Which type of chromatography would be most effective for separating a protein with a His-tag from a complex mixture?

Accepted Answer

Affinity chromatography using a nickel or cobalt resin

Question 5

A protein mixture is loaded onto a cation-exchange column at pH 7.0. Protein A has a net charge of +2, and Protein B has a net charge of -1. Which protein will elute first?

Accepted Answer

Protein B, because it does not bind to the negatively charged resin and elutes first.

Question 6

In size-exclusion (gel filtration) chromatography, which protein will elute first from the column?

Accepted Answer

The largest protein, because it is excluded from the pores and travels a shorter path.

Question 7

A purification table shows the following data for an enzyme: initial specific activity = 10 units/mg, final specific activity = 600 units/mg. What is the purification factor?

Accepted Answer

$$\frac{600}{10} = 60$$

Question 8

Which of the following best describes the principle of SDS-PAGE for protein separation?

Accepted Answer

Proteins are separated based on their size, as SDS binds uniformly and imparts a negative charge proportional to mass.

Question 9

A researcher wants to separate proteins based on their isoelectric point (pI). Which technique should be used?

Accepted Answer

Isoelectric focusing

Question 10

In two-dimensional gel electrophoresis, proteins are first separated by isoelectric focusing and then by SDS-PAGE. What is the advantage of this method?

Accepted Answer

It separates proteins by both their isoelectric point and molecular weight, increasing resolution.

Question 11

Which of the following statements about ion-exchange chromatography is correct?

Accepted Answer

Anion exchangers bind negatively charged proteins.

Question 12

A protein sample is analyzed by SDS-PAGE and stained with Coomassie Blue. What information can be obtained from this experiment?

Accepted Answer

The approximate molecular weight and purity of the protein.

Question 13

Which of the following is a strong cation exchanger used in ion-exchange chromatography?

Accepted Answer

SP (Sulphopropyl)

Question 14

A protein with a pI of 5.0 is placed in a buffer at pH 7.5 and loaded onto a DEAE column. What will happen?

Accepted Answer

The protein will have a net negative charge and bind to the column.

Question 15

Which purification step is most likely to result in the greatest increase in specific activity for an enzyme?

Accepted Answer

Affinity chromatography

Question 16

A scientist is comparing the proteomes of healthy and cancerous cells using 2D gel electrophoresis. What does the appearance of a blue spot unique to the cancer cell sample indicate?

Accepted Answer

A protein present only in cancer cells.

Question 17

Which of the following best describes the function of the mobile phase in column chromatography?

Accepted Answer

It carries the protein sample through the column, allowing separation based on interactions with the stationary phase.

Question 18

In affinity chromatography, how is the protein of interest typically eluted from the column?

Accepted Answer

By adding a solution containing a free ligand that competes for binding with the immobilized ligand.

Question 19

Which of the following is a key advantage of recombinant protein expression in bacteria compared to purification from animal tissues?

Accepted Answer

Recombinant expression allows for higher yields and easier purification of the target protein.

Protein Purification and Characterization Techniques

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