Question 1

Why is protein purification considered a core skill in biochemistry, and what is one major consequence of failing to purify a protein before studying its function?

Accepted Answer

It allows for the study of protein function without interference from other cellular components; failing to purify can result in misleading results due to contaminating proteins.

Question 2

A biochemist wants to isolate cytochrome C from heart mitochondria. Which initial step is most important for maximizing yield and purity?

Accepted Answer

Selecting a rich source tissue, such as heart mitochondria, to ensure high abundance of cytochrome C.

Question 3

Which method is most appropriate for lysing animal cells to release soluble proteins?

Accepted Answer

Hypotonic shock, which causes cells to swell and rupture due to osmotic imbalance.

Question 4

A protein sample is subjected to differential centrifugation. Which cellular component will be found in the pellet after spinning at low RPM?

Accepted Answer

Mitochondria, due to their higher density compared to soluble proteins.

Question 5

Which equation describes the relationship between absorbance and concentration in spectrophotometric analysis?

Accepted Answer

$$A = \epsilon b c$$

Question 6

During protein purification, what does an increase in specific activity indicate?

Accepted Answer

The protein sample is becoming more pure, with fewer contaminating proteins.

Question 7

Which chromatography method would you use to separate proteins based on their net charge at a given pH?

Accepted Answer

Ion exchange chromatography

Question 8

A protein has a pI of 8.5. If you use a cation exchange resin at pH 7.0, what will happen?

Accepted Answer

The protein will carry a net positive charge and bind to the negatively charged resin.

Question 9

Which salt is commonly used for 'salting out' proteins during purification, and why?

Accepted Answer

$$(NH_4$)_2SO_4 is $$used because it is highly soluble and does not harm proteins.

Question 10

In gel filtration chromatography, which protein will elute first: a 150 kDa protein or a 20 kDa protein?

Accepted Answer

The 150 kDa protein, because larger proteins are excluded from the gel pores and travel faster.

Question 11

Which principle allows affinity chromatography to specifically purify a target protein?

Accepted Answer

Molecular complementarity between the protein and a ligand attached to the matrix.

Question 12

A purification table shows a decrease in total activity but an increase in specific activity after several steps. What does this suggest?

Accepted Answer

Some enzyme was lost, but the remaining sample is more pure.

Question 13

Which method separates proteins based on both charge and size, and is commonly visualized with Coomassie blue staining?

Accepted Answer

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)

Question 14

What is the effect of SDS on proteins during SDS-PAGE?

Accepted Answer

SDS denatures proteins and gives them a uniform negative charge, allowing separation by molecular mass.

Question 15

In isoelectric focusing, where will a protein with pI = 6 migrate in a pH gradient from 3 to 10?

Accepted Answer

It will migrate until it reaches the region where pH = 6, then stop.

Question 16

A protein sample is dialyzed against a buffer. What is the main purpose of dialysis in protein purification?

Accepted Answer

To remove small molecules and salts while retaining the protein inside the dialysis bag.

Question 17

Which equation describes the force on an ion in an electric field during electrophoresis?

Accepted Answer

$$F_{ele} = qE$$

Question 18

In paper chromatography, what determines how far a molecule migrates?

Accepted Answer

Its partition coefficient between the mobile and stationary phases.

Question 19

Which visualization method is most sensitive for detecting proteins after electrophoresis?

Accepted Answer

Silver staining

Question 20

A protein is purified using affinity chromatography with a ligand attached to agarose beads. How is the protein typically eluted from the column?

Accepted Answer

By adding excess free ligand to compete for binding and release the protein.

Protein Purification and Analytical Techniques in Biochemistry

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