Q: Which equation correctly defines specific activity in a protein purification experiment?

$$\text{Specific Activity} = \frac{\text{Total Activity (U)}}{\text{Total Protein (mg)}}$$

Question 1

Which of the following best explains why proteins must be kept under specific conditions during purification to preserve their function?

Accepted Answer

Proteins can denature or lose activity if exposed to inappropriate pH, temperature, or solvents, making functional assays impossible.

Question 2

During ion exchange chromatography, a protein with a pI of 5.5 is loaded onto a cation exchange column at pH 7.5. What is the net charge of the protein, and what is the expected behavior?

Accepted Answer

The protein is negatively charged and will elute quickly from the column.

Question 3

Which equation correctly defines specific activity in a protein purification experiment?

Accepted Answer

$$	ext{Specific Activity} = \frac{	ext{Total Activity (U)}}{	ext{Total Protein (mg)}}$$

Question 4

A protein sample is analyzed by SDS-PAGE and shows a single band at 40 kDa, but size exclusion chromatography (SEC) indicates a native molecular weight of 120 kDa. What is the most likely explanation?

Accepted Answer

The protein is a trimer of 40 kDa subunits.

Question 5

Which of the following best describes the principle behind size exclusion chromatography?

Accepted Answer

Proteins are separated based on their hydrodynamic size, with larger proteins eluting first.

Question 6

In the Bradford assay for protein quantification, what is the role of Coomassie dye?

Accepted Answer

It binds to proteins, causing a shift in absorbance that can be measured at 595 nm.

Question 7

Why is a salt gradient used in ion exchange chromatography rather than a fixed high-salt buffer?

Accepted Answer

A salt gradient allows for sequential elution of proteins based on their strength of interaction with the column, improving resolution.

Question 8

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

Accepted Answer

SDS-PAGE separates proteins based on size, with SDS providing a uniform negative charge to all proteins.

Question 9

A protein is purified using affinity chromatography with a ligand-coupled resin. Which risk is unique to this approach compared to IMAC (His-tag) purification?

Accepted Answer

The ligand may be chemically altered during coupling, reducing its ability to bind the protein.

Question 10

Which of the following best describes the process of isoelectric focusing?

Accepted Answer

Proteins migrate through a pH gradient until they reach a region where their net charge is zero (their pI).

Question 11

Which of the following correctly describes the cleavage specificity of trypsin?

Accepted Answer

Trypsin cleaves on the C-terminal side of basic residues (K, R), unless followed by proline.

Question 12

In electrospray ionization mass spectrometry (ESI-MS), why do proteins often appear as multiple peaks with different m/z values?

Accepted Answer

Proteins acquire multiple charges, resulting in different m/z values for the same molecular weight.

Question 13

A researcher uses differential centrifugation to fractionate a cell homogenate. Which component is most likely to pellet at 1000 x g for 10 minutes?

Accepted Answer

Nuclei

Question 14

Which of the following best explains why 2D-gel electrophoresis provides higher resolution than either SDS-PAGE or isoelectric focusing alone?

Accepted Answer

It separates proteins by both isoelectric point and size, reducing the chance that two proteins will co-migrate.

Question 15

Which of the following is a correct statement about the use of NADH in enzyme assays for lactate dehydrogenase?

Accepted Answer

NADH is fluorescent and its decrease can be monitored to assay enzyme activity.

Question 16

Which of the following best describes the main difference between in vitro and in vivo studies of protein function?

Accepted Answer

In vitro studies are performed with isolated proteins in controlled environments, while in vivo studies are performed in living organisms.

Protein Purification and Analytical Techniques in Biochemistry

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