Q: If a protein is in a region of the IEF gel where $pH < pI$, what is the net charge of the protein?

The protein is positively charged.

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

Toward the anode (positive electrode)

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

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

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

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

Q: 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.

The tripeptide will focus near $pH = 3.0$

Q: Which of the following statements about isoelectric focusing is FALSE?

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

Q: 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?

Net positive charge; migrates toward the cathode.

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

The pH gradient is very stable and finely controlled.

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 $pH < pI$, 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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