StudyStore

Free MCAT-style practice, 1A

MCAT Protein Purification, SDS-PAGE and Chromatography Practice Questions

By StudyStore · Published 2026-11-04 · 8 original questions · about 12 min

Eight free MCAT-style practice questions on SDS-PAGE, native PAGE, isoelectric focusing, size exclusion, ion exchange and affinity chromatography, with explanations.

Email me free MCAT notes, practice sets and new packs from StudyStore. Double opt-in, unsubscribe in one click. Privacy

Quick concept primer

Protein separation questions ask what property a technique sorts by. SDS-PAGE uses the detergent SDS, which denatures proteins and coats them with negative charge in proportion to length, so all proteins run toward the anode and separate by mass alone: smaller proteins migrate farther. Adding a reducing agent such as beta-mercaptoethanol breaks disulfide bonds and separates subunits. Native PAGE keeps folding and charge, so migration depends on charge, size and shape. Isoelectric focusing separates proteins in a pH gradient until each reaches its pI, where net charge is zero.

Column methods: size exclusion (gel filtration) lets small molecules enter beads, so large proteins elute first. Ion exchange binds proteins of opposite charge to the resin and elutes them with rising salt or a pH change. Affinity chromatography uses a specific ligand, such as a His-tag bound to nickel, so the target sticks and everything else washes through.

8 practice questions

Try each question before opening the explanation. Click "Show answer and explanation" to check yourself. All questions are original and written for study practice.

Question 1

In SDS-PAGE, proteins separate mainly according to which property?

  1. Isoelectric point
  2. Molecular mass
  3. Binding affinity for a ligand
  4. Hydrophobicity of the side chains
Show answer and explanation

Answer: B. Molecular mass

SDS denatures proteins and gives them a roughly uniform negative charge per unit mass, so charge to mass is nearly constant and the gel sieves them by size. Isoelectric point is the basis of isoelectric focusing, and ligand affinity is the basis of affinity chromatography.

Question 2

A protein of 25 kDa and a protein of 100 kDa are run on an SDS-PAGE gel. Which statement is correct?

  1. The 100 kDa protein migrates farther toward the anode
  2. The two proteins migrate the same distance because both are coated with SDS
  3. The 25 kDa protein migrates toward the cathode
  4. The 25 kDa protein migrates farther toward the anode
Show answer and explanation

Answer: D. The 25 kDa protein migrates farther toward the anode

SDS-coated proteins are negatively charged and move toward the positive electrode (anode). The gel matrix slows large proteins more, so the smaller 25 kDa protein travels farther. Equal charge density does not mean equal migration, because the gel acts as a sieve.

Question 3

A protein runs as a single band at 60 kDa on SDS-PAGE without a reducing agent, but as two bands at 35 kDa and 25 kDa when beta-mercaptoethanol is added. What is the most likely explanation?

  1. The protein has two subunits joined by a disulfide bond
  2. The protein is a dimer held together only by hydrogen bonds
  3. The protein was degraded by the reducing agent
  4. The protein has two different pI values
Show answer and explanation

Answer: A. The protein has two subunits joined by a disulfide bond

Beta-mercaptoethanol reduces disulfide bonds. SDS alone disrupts noncovalent interactions such as hydrogen bonds, so a band that splits only after adding a reducing agent indicates subunits linked by a covalent disulfide. The masses also add up: 35 plus 25 equals 60 kDa.

Question 4

In a size exclusion column, which protein elutes first?

  1. The smallest protein, because it passes through the beads fastest
  2. The most positively charged protein
  3. The largest protein, because it is excluded from the bead pores
  4. The protein with the highest pI
Show answer and explanation

Answer: C. The largest protein, because it is excluded from the bead pores

Large proteins cannot enter the pores of the beads and take the shorter path around them, so they elute first. Small proteins wander into the pores and take longer. Charge and pI do not matter in an ideal size exclusion separation.

Question 5

A protein with a pI of 9.0 is applied to a cation exchange column, which has negatively charged resin, at pH 7.0. What happens?

  1. The protein binds because it carries a net positive charge at pH 7.0
  2. The protein binds because it is neutral at pH 7.0
  3. The protein flows through because it is positively charged
  4. The protein flows through because it is hydrophobic
Show answer and explanation

Answer: A. The protein binds because it carries a net positive charge at pH 7.0

When pH is below pI, the protein carries a net positive charge. A positively charged protein binds the negatively charged resin of a cation exchanger. It is then eluted with increasing salt concentration, which competes for the resin, or by raising the pH toward its pI.

Question 6

In isoelectric focusing, a protein with pI 6.5 is placed at a spot in the gradient where pH is 8.0. In which direction does it move?

  1. Toward the cathode, because it is positively charged
  2. Toward the anode, because it is negatively charged
  3. It does not move because it is in a pH gradient
  4. Toward the anode, because it is positively charged
Show answer and explanation

Answer: B. Toward the anode, because it is negatively charged

At pH 8.0, which is above the pI of 6.5, the protein is deprotonated and carries a net negative charge, so it moves toward the anode. As it moves to lower pH, it gains protons and its charge falls to zero at pH 6.5, where it stops.

Question 7

A researcher adds a polyhistidine tag to a recombinant protein and passes a cell lysate over a column containing immobilized nickel. This technique is best described as

  1. size exclusion chromatography
  2. affinity chromatography
  3. anion exchange chromatography
  4. native PAGE
Show answer and explanation

Answer: B. affinity chromatography

The histidine tag binds nickel specifically, so only the tagged protein is retained while other proteins wash through. This ligand-specific retention defines affinity chromatography. The target is later eluted with imidazole, which competes for the nickel.

Question 8

Why might a protein with a small mass but a large net negative charge migrate differently on native PAGE than a larger, less charged protein, compared with SDS-PAGE?

  1. Native PAGE denatures both proteins before separation
  2. Native PAGE separates strictly by mass, like SDS-PAGE
  3. Native PAGE uses a pH gradient so both proteins stop at their pI
  4. Native PAGE preserves intrinsic charge and shape, so charge influences migration
Show answer and explanation

Answer: D. Native PAGE preserves intrinsic charge and shape, so charge influences migration

Native PAGE does not denature the protein, so migration depends on intrinsic net charge, size and three-dimensional shape together. A highly charged small protein can outrun a bigger, less charged one. SDS-PAGE removes these differences by giving all proteins similar charge density, so mass dominates.

Free MCAT sampler

Free sample: MCAT cheat sheet (PDF)

Join the list and download a free sample from a paid pack: the 3-page Amino Acids, Proteins and Enzymes cheat sheet (PDF). We also email free MCAT notes and practice sets.

Free sampler, no signup needed: 51 study notes with original diagrams and 5-question quizzes, 80 practice sets (706 questions with full explanations) and the 2027 study schedule generator.

Email me free MCAT notes, practice sets and new packs from StudyStore. Double opt-in, unsubscribe in one click. Privacy

Keep practicing

Written by StudyStore from standard scientific facts as a study aid. These are not official exam questions and are not affiliated with or endorsed by any testing organization. Always check values against the reference provided with your exam or course.

Spotted a mistake in a question? Report a problem or suggest something