Question 1

The following methods are useful for cell homogenization:

Accepted Answer

All the methods listed (sonication, freezing and thawing, detergents, and enzymes) are commonly used techniques for cell homogenization, each working through different mechanisms to break down cell structures and membranes to release cellular components.

Question 2

The purity of an enzyme at various stages of purification is best measured by

Accepted Answer

Specific activity of the enzyme, which is the ratio of enzyme activity to the amount of protein in the sample, is the best measure of enzyme purity. It increases as the enzyme becomes purer through the purification process.

Question 3

The typical order for the major steps of enzyme isolation would be (from first to last):

Accepted Answer

Homogenization is typically the first step to break open cells and release their contents. Salt fractionation is then used to precipitate proteins out of solution at different salt concentrations. Column chromatography further purifies the enzyme based on size, charge, or binding affinity. Electrophoresis can be used as a final step to analyze the purity of the enzyme.

Question 4

Which of the following are principles on which to base column chromatography?

Accepted Answer

Column chromatography can be based on various principles including molecular size, isoionic pH or pI, and ion exchange, making all the options correct.

Question 5

Which separates based on the ionic charge on a protein?

Accepted Answer

Cation exchange chromatography separates molecules based on their net positive charge, while anion exchange chromatography separates molecules based on their net negative charge. Both techniques separate based on the ionic charge on a protein.

Question 6

In gel filtration chromatography

Accepted Answer

In gel filtration chromatography, also known as size exclusion chromatography, materials are separated based on their size, with the larger molecules eluting first because they are excluded from the pores of the gel and therefore travel more directly through the column. Smaller molecules enter the pores, which delays their elution.

Question 7

Differential centrifugation at low speeds (several thousand RPM) is a useful step when

Accepted Answer

Differential centrifugation at low speeds is used to separate larger components like organelles or debris from a mixture based on size and density.

Question 8

Using differential centrifugation it is possible to separate

Accepted Answer

Differential centrifugation separates particles based on their size and density. Nuclei, mitochondria, and ribosomes have different sizes and densities, so they can be separated into three separate fractions using differential centrifugation.

Question 9

Which would be best to separate proteins of similar size?

Accepted Answer

Cation or anion exchange chromatography can separate proteins based on their charge differences, which is effective even for proteins of similar sizes.

Question 10

Which of the following happens as a protein is purified?

Accepted Answer

During the purification process, the percent recovery typically decreases because some amount of protein is lost at each purification step. However, the fold purification increases because the protein becomes more concentrated relative to other proteins and contaminants.

Question 11

In affinity chromatography, a protein

Accepted Answer

In affinity chromatography, proteins that have a specific binding affinity for the ligand attached to the chromatography matrix will bind to the ligand and remain on the column. Other proteins, which do not have this specific affinity, will elute from the column.

Question 12

Which type of column is most affected by the shape of the protein, for example, comparing spherical and cigar-shaped proteins?

Accepted Answer

Gel filtration chromatography separates molecules based on their size and shape. Spherical proteins will navigate through the matrix differently than elongated, cigar-shaped proteins, affecting their elution times. This method is particularly sensitive to the physical dimensions of the proteins being separated.

Question 13

Ammonium sulfate is useful in protein purification because

Accepted Answer

Ammonium sulfate is used in protein purification primarily because it reduces the solubility of proteins in solution. This is achieved through the mechanism of "salting out," where the high concentration of ammonium sulfate increases the ionic strength of the solution. This, in turn, disrupts the water-protein interactions (via ion-dipole interactions) and makes the proteins less soluble, causing them to precipitate out of the solution. This method does not rely on the sparing solubility of ammonium sulfate itself (as suggested in option B) nor directly on its nitrogen and sulfur content (as suggested in option A). The purity of proteins obtained (option C) is a result of the precipitation process but is not the primary reason why ammonium sulfate is used.

Question 14

Elution of proteins by means of a pH gradient would work best with this type of column:

Accepted Answer

Elution of proteins by means of a pH gradient works best with ion exchange chromatography, which includes both cation and anion exchange columns. This method separates proteins based on their charge, which can be manipulated by changing the pH.

Question 15

Which would be best to separate a protein that binds strongly to its substrate?

Accepted Answer

Affinity chromatography is the best method for separating a protein that binds strongly to its substrate because it utilizes a specific interaction between the protein and a ligand that is immobilized on the chromatography matrix, allowing for highly selective separation.

Question 16

The typical order of differential centrifugation for organelles is (from slowest speed\lowest g to fastest speed\highest g ):

Accepted Answer

Differential centrifugation separates cell components by size and density at increasing speeds. The correct order starts with whole cells (largest and least dense), followed by nuclei, then mitochondria and chloroplasts (similar in size), next microsomes (smaller vesicles), and finally the cytosol (soluble fraction).

Question 17

What tends to happen to the percent recovery during a protein's purification?

Accepted Answer

The percent recovery usually steadily decreases during the purification process because some amount of the protein is lost at each purification step.

Question 18

Salting out with ammonium sulfate is based upon proteins interacting with other proteins via

Accepted Answer

Salting out with ammonium sulfate reduces the solubility of proteins primarily through the promotion of hydrophobic interactions among protein molecules, leading to their precipitation. This process decreases the availability of water molecules for protein solvation by increasing the ionic strength of the solution.

Question 19

Which would be best to separate positively charged proteins?

Accepted Answer

Cation exchange chromatography is best for separating positively charged proteins because it uses a negatively charged resin to attract and bind the positively charged molecules.

Question 20

Which separates on the basis of molecular weight?

Accepted Answer

Gel filtration chromatography separates molecules based on their size or molecular weight, with smaller molecules eluting later as they are retained within the porous matrix of the gel.

Quiz 5: Protein Purification and Characterization Techniques