Question 1

Which property of proteins is exploited during isoelectric focusing (IEF) to achieve separation?

Accepted Answer

Their net charge at different pH values

Question 2

During isoelectric focusing, what happens to a protein when it reaches the region of the gel where the pH equals its isoelectric point ($$pI$$)?

Accepted Answer

It stops moving because its net charge is zero

Question 3

A protein with a $$pI of 5.5 is $$placed in an isoelectric focusing gel with a pH gradient from 3 to 9. Toward which electrode will the protein initially migrate if loaded at pH 7?

Accepted Answer

Toward the anode (positive electrode)

Question 4

Which of the following best describes the pH gradient in an isoelectric focusing gel?

Accepted Answer

The pH changes gradually from one end of the gel to the other

Question 5

A peptide has a $$pI of 4.2. In an $$IEF gel with a pH gradient from 3 to 7, at which pH will the peptide stop migrating?

Accepted Answer

$$pH = 4.2$$

Question 6

If a protein is loaded into an IEF gel at a position where the pH is lower than its $$pI$$, what will be its net charge and direction of migration?

Accepted Answer

It will be positively charged and migrate toward higher pH

Question 7

Which of the following statements about isoelectric focusing is FALSE?

Accepted Answer

Proteins with the same $$pI$$ but different molecular weights will separate from each other

Question 8

Given two peptides, Peptide #1 ($$MW = 200$$, $$pI = 4.0) $$and Peptide #2 ($$MW = 400$$, $$pI = 7.0$$), which will migrate farther in an IEF gel with a pH gradient from 3 to 9?

Accepted Answer

Peptide #2, because it has a higher $$pI$$

Question 9

A tripeptide composed of Glu-Met-Asp has the following $$pK_a$$ values: $$pK_{a,1} = 2.1$$, $$pK_{a,2} = 3.9$$, $$pK_{a,3} = 9.7. $$What is the approximate isoelectric point ($$pI) of $$this peptide?

Accepted Answer

$$pI = \frac{2.1 + 3.9}{2} = 3.0$$

Question 10

Why do proteins stop migrating in an IEF gel when they reach their isoelectric point?

Accepted Answer

Their net charge becomes zero, so they no longer respond to the electric field

Question 11

A protein with a $$pI of 8.0 is $$loaded into an IEF gel at pH 6.0. What is its net charge and migration direction?

Accepted Answer

Net positive charge; migrates toward higher pH

Question 12

Which of the following best explains why isoelectric focusing can separate proteins with similar molecular weights?

Accepted Answer

Proteins are separated based on their $$pI$$, not their size

Question 13

During isoelectric focusing, what would happen if the pH gradient was not properly established in the gel?

Accepted Answer

Proteins would not separate based on $$pI$$

Question 14

A protein with a $$pI of 6.5 is $$loaded into an IEF gel at pH 8.0. What is its net charge and migration direction?

Accepted Answer

Net negative charge; migrates toward lower pH

Question 15

Which of the following is TRUE regarding the movement of proteins in an IEF gel?

Accepted Answer

Proteins with net positive charge migrate toward higher pH

Question 16

If a protein has a $$pI of 5.0 $$and is placed in a region of the gel with pH 5.0, what is its net charge?

Accepted Answer

Zero

Question 17

A mixture of proteins is separated by IEF. Which protein will be found closest to the anode (positive electrode) after separation?

Accepted Answer

The protein with the lowest $$pI$$

Question 18

Which of the following is a necessary condition for successful isoelectric focusing?

Accepted Answer

A stable pH gradient across the gel

Question 19

A protein with a $$pI of 7.4 is $$loaded into an IEF gel at pH 7.4. What will happen?

Accepted Answer

It will not migrate

Question 20

Which of the following best describes the principle behind isoelectric focusing?

Accepted Answer

Proteins are separated based on their net charge at different pH values

Isoelectric Focusing: Principles and Applications in Protein Separation

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