Question 1

Which principle is the basis for protein separation in isoelectric focusing (IEF)?

Accepted Answer

Proteins are separated based on their net charge at a specific pH.

Question 2

During isoelectric focusing, what happens to a protein when it reaches its isoelectric point ($$pI$$)?

Accepted Answer

It has no net charge and stops migrating in the gel.

Question 3

A protein with a $$pI of 5.5 is $$placed in an IEF gel with a pH gradient from 3 to 9. At which pH will the protein stop moving?

Accepted Answer

$$pH = 5.5$$

Question 4

If a protein is in a region of the IEF gel where $$pH \u003c pI$$, what is the net charge of the protein?

Accepted Answer

The protein is positively charged.

Question 5

Which direction will a protein with a net negative charge migrate in an IEF gel?

Accepted Answer

Toward the anode (positive electrode)

Question 6

A peptide has a $$pI of 8.0. In an $$IEF gel with a pH gradient from 4 to 10, where will the peptide be found after focusing?

Accepted Answer

At the region where $$pH = 8$$

Question 7

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

Accepted Answer

The pH increases gradually from one end of the gel to the other.

Question 8

A mixture contains two peptides: Peptide A ($$pI = 4.5) $$and Peptide B ($$pI = 7.2). In an $$IEF gel with a pH gradient from 3 to 9, which peptide will be closer to the anode after focusing?

Accepted Answer

Peptide A

Question 9

During isoelectric focusing, why do proteins stop migrating when they reach their $$pI$$?

Accepted Answer

They have no net charge and thus do not respond to the electric field.

Question 10

A tripeptide composed of Glu-Met-Asp is analyzed by IEF. Given the $$pI$$ values: Glu ($$pI = 3.2$$), Met ($$pI = 5.7$$), Asp ($$pI = 2.8$$), estimate the $$pI of $$the tripeptide and predict its position in a gel with a pH gradient from 3 to 9.

Accepted Answer

The tripeptide will focus near $$pH = 3.0$$

Question 11

Which of the following statements about isoelectric focusing is FALSE?

Accepted Answer

Proteins with the same $$pI$$ can be separated by IEF.

Question 12

In a clinical laboratory, a patient’s serum protein is analyzed by IEF. The protein focuses at $$pH = 6.8. $$What does this indicate about the protein’s $$pI$$?

Accepted Answer

The protein’s $$pI is 6.8.$$

Question 13

A protein with a $$pI of 9.0 is $$placed in a region of the gel with $$pH = 7.0. $$What is the net charge of the protein and in which direction will it migrate?

Accepted Answer

Net positive charge; migrates toward the cathode.

Question 14

Which of the following best explains why isoelectric focusing can resolve proteins that differ by only 0.01 pH units in their $$pI$$?

Accepted Answer

The pH gradient is very stable and finely controlled.

Question 15

A mixture of three peptides is analyzed by IEF: Peptide 1 ($$pI = 4.0$$), Peptide 2 ($$pI = 7.0$$), Peptide 3 ($$pI = 9.0). $$Which peptide will migrate the farthest from the application point if the sample is loaded at $$pH = 6.0$$?

Accepted Answer

Peptide 3

Question 16

In 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 17

A protein is loaded into an IEF gel at $$pH = 8.0$$ and has a $$pI = 6.0. $$What is the initial net charge of the protein and in which direction will it migrate?

Accepted Answer

Net negative charge; migrates toward the anode.

Question 18

Which of the following equations is used to estimate the isoelectric point ($$pI) of a $$simple peptide with two ionizable groups?

Accepted Answer

$$pI = \frac{pK_a + pK_b}{2}$$

Question 19

A protein with a $$pI of 5.0 is $$placed in a gel with a pH gradient from 3 to 9. If the protein is initially loaded at $$pH = 7.0$$, describe its migration.

Accepted Answer

It will migrate toward lower pH until it reaches $$pH = 5.0.$$

Isoelectric Focusing: Principles and Applications in Protein Separation

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