Question 1

Which statement best explains the partial double bond character of the peptide bond as shown in the resonance structures above?

Accepted Answer

The peptide bond is formed by the resonance between the lone pair on the nitrogen and the carbonyl carbon, resulting in partial double bond character.

Question 2

Given the following pKa values for ionizable groups in polypeptides: C-terminus (2.0), N-terminus (9.5), Asp (3.9), Glu (4.1), His (6.0), Cys (8.4), Tyr (10.5), Lys (10.5), Arg (12.5), what is the net charge of the peptide sequence E-K-H at pH 7?

Accepted Answer

$$+1$$

Question 3

A protein has a pI of 5.0. At pH 7.0, what will be the net charge of the protein and how will it behave in an anion exchange chromatography column?

Accepted Answer

The protein will be negatively charged and bind to the column.

Question 4

Which of the following best describes the effect of SDS in SDS-PAGE?

Accepted Answer

SDS coats proteins with a uniform negative charge and denatures them, so migration depends only on size.

Question 5

You have a mixture of three proteins: 15 kDa, 30 kDa, and 75 kDa. Which protein will elute first from a size exclusion chromatography column?

Accepted Answer

The 75 kDa protein

Question 6

A peptide has the sequence D-E-K-R. Using the pKa values: Asp (3.9), Glu (4.1), Lys (10.5), Arg (12.5), N-term (9.5), C-term (2.0), what is the net charge at pH 7?

Accepted Answer

$$+1$$

Question 7

Which method would you use to demonstrate that two proteins (15 kDa and 30 kDa) form a dimer in solution?

Accepted Answer

Run native PAGE and look for a band at 45 kDa.

Question 8

Why might you use Western blotting instead of Coomassie Blue staining after SDS-PAGE?

Accepted Answer

Western blotting is more sensitive and can detect a specific protein among many.

Question 9

Which of the following best describes the elution of a His-tagged protein from a nickel affinity column?

Accepted Answer

Elution with imidazole, which competes with the His-tag for binding to nickel.

Question 10

A protein mixture is loaded onto a cation exchange column at pH 6.0. Which protein will bind most tightly?

Accepted Answer

The protein with the highest pI

Question 11

What is the main reason that proteins migrate at different rates in native PAGE?

Accepted Answer

Differences in charge, size, and shape of the native proteins.

Question 12

If you want to prevent two proteins from interacting in solution, would you use a high salt or low salt buffer?

Accepted Answer

High salt buffer to disrupt electrostatic interactions.

Question 13

Which of the following is true about the isoelectric point (pI) of a protein?

Accepted Answer

At pH = pI, the protein has no net charge and is least soluble.

Question 14

Which of the following best describes the effect of DTT or β-mercaptoethanol in SDS-PAGE sample preparation?

Accepted Answer

They reduce disulfide bonds, allowing proteins to fully denature.

Question 15

A protein with a pI of 9.5 is placed in a buffer at pH 7.0. What is its net charge and how will it behave in cation exchange chromatography?

Accepted Answer

It will be positively charged and bind to the column.

Question 16

Which of the following best explains why proteins with similar molecular weights but different shapes may migrate differently in native PAGE?

Accepted Answer

Native PAGE separates proteins based on charge, size, and shape.

Question 17

You are purifying a protein with a His-tag using immobilized metal affinity chromatography (IMAC). Which elution method would you use?

Accepted Answer

Elution with imidazole, which competes with the His-tag for binding to the metal ion.

Question 18

A peptide has the sequence H-E-K. Using the pKa values: His (6.0), Glu (4.1), Lys (10.5), N-term (9.5), C-term (2.0), what is the net charge at pH 6.0?

Accepted Answer

$$+1$$

Question 19

Which of the following best describes the principle of affinity chromatography?

Accepted Answer

Separation based on specific binding interactions between the protein and a ligand attached to the matrix.

Amino Acids, Peptide Bonds, and Protein Purification: Study Notes for Biochemistry

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