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📝 Organelle isolation by cell fractionation (8 MCQs)

📖 From Principles of Biochemistry • 1. The Foundations of Biochemistry • 8 questions available

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What is Organelle isolation by cell fractionation?

Definition:
Organelle isolation by cell fractionation is a laboratory technique that separates cellular organelles based on their physical and chemical properties, including size, shape, density, and sedimentation rate, by first disrupting the cell membrane through homogenization, then using differential and density gradient centrifugation to obtain enriched fractions of nuclei, mitochondria, lysosomes, peroxisomes, and other organelles for biochemical and functional studies.

Working:
This procedure works by breaking open cells in a buffer to release organelles, then centrifuging the homogenate at progressively higher speeds: low speed (1000g) pellets nuclei and unbroken cells, medium speed (10,000g) pellets mitochondria and lysosomes, high speed (100,000g) pellets microsomes, and ultracentrifugation with density gradients can separate organelles with similar sedimentation rates, with the centrifugal force calculated by $F_c = m \omega^2 r$ , where $m$ is mass, $\omega$ is angular velocity, and $r$ is rotor radius.

Example:
A simple example is isolating mitochondria from rat liver, where the tissue is homogenized in a cold sucrose buffer, centrifuged at 600g for 10 minutes to remove nuclei, then the supernatant is spun at 10,000g for 15 minutes to pellet mitochondria, which are then washed and resuspended for measuring oxygen consumption or enzyme activity.

Reason:
Organelle isolation is fundamental for studying cellular processes, as it allows researchers to analyze the function, composition, and biochemistry of individual organelles, enabling discoveries in metabolism, cell signaling, and disease mechanisms, and is crucial in drug development and diagnostics.

Easy

Medium

Hard

📝 All Organelle isolation by cell fractionation MCQs

Q1. A cell homogenate contains nuclei, mitochondria, lysosomes, and soluble proteins. If the goal is to obtain a fraction highly enriched in nuclei before studying smaller organelles, which strategy is most appropriate?

A.Use progressively lower centrifugal speeds to pellet smaller particles first

B.Use a relatively low centrifugal speed first, collect the pellet, then analyze the supernatant ✅

C.Use isopycnic centrifugation immediately and discard all material that reaches the bottom

D.Increase centrifugation speed continuously without separating fractions

💡 Difficulty: medium | ✅ Correct: B

📖 Explanation: A relatively low centrifugal speed preferentially pellets larger and denser structures such as nuclei. The remaining supernatant retains smaller organelles and soluble components, allowing sequential fractionation and reducing contamination in later fractions.

Q2. Two organelles have similar sizes but different buoyant densities. A researcher wants to separate them even though size-based differential centrifugation gives substantial overlap. Which method is most logically advantageous?

A.Differential centrifugation because equal-sized particles always sediment together

B.Isopycnic centrifugation because particles migrate toward positions determined mainly by buoyant density ✅

C.Homogenization because it increases differences in organelle density

D.Using a shorter centrifugation time because density differences disappear with time

💡 Difficulty: hard | ✅ Correct: B

📖 Explanation: Differential centrifugation primarily exploits differences in sedimentation behavior, which depends strongly on size and mass. Isopycnic centrifugation is better when particles have sufficiently different buoyant densities because each moves toward its density-equilibrium position.

Q3. A researcher performs differential centrifugation and obtains a pellet after a low-speed spin. The supernatant is then centrifuged at a higher speed. What does the second pellet most likely contain relative to the first pellet?

A.Mostly larger structures that sedimented even more slowly

B.Mostly smaller organelles that remained suspended during the first spin ✅

C.Only soluble proteins because proteins sediment faster than organelles

D.Exactly the same composition as the first pellet

💡 Difficulty: medium | ✅ Correct: B

📖 Explanation: The first low-speed step removes structures that sediment readily, particularly larger components. Increasing the speed causes smaller organelles that remained in the supernatant to sediment. Therefore, the second pellet is enriched in smaller structures but may still contain contaminants.

Q4. A laboratory reports that its mitochondrial fraction contains many lysosomes. The team used differential centrifugation but increased the speed so much during the first separation that several smaller organelles also pelleted. What is the most likely procedural error?

A.The first centrifugation was too gentle to sediment any particles

B.The initial centrifugation was too forceful, causing premature sedimentation of smaller organelles ✅

C.Isopycnic centrifugation always destroys lysosomes

D.Homogenization completely prevents mitochondrial sedimentation

💡 Difficulty: hard | ✅ Correct: B

📖 Explanation: In differential centrifugation, increasing centrifugal force causes progressively smaller particles to sediment. If the initial spin is too strong, smaller organelles such as lysosomes can pellet with larger structures, lowering the purity of the mitochondrial fraction.

Q5. A graph shows the percentage of an organelle recovered in successive pellets as centrifugal speed increases: Pellet 1 at low speed = 70%, Pellet 2 at medium speed = 20%, Pellet 3 at high speed = 8%, soluble fraction = 2%. Which interpretation is best supported?

A.Most of the organelle sediments during the earliest centrifugation step ✅

B.The organelle remains soluble until the highest speed

C.The organelle is evenly distributed among all fractions

D.The data prove that the organelle has the highest buoyant density in the sample

💡 Difficulty: medium | ✅ Correct: A

📖 Explanation: The recovery data show that 70% of the organelle appears in the first pellet, indicating that most of it sediments under the lowest tested speed. The distribution across later fractions represents incomplete separation rather than proof of buoyant density.

Q6. A researcher wants to compare two isolation methods. Method X produces a mitochondrial fraction with 85% mitochondrial markers but only 55% recovery. Method Y produces 60% mitochondrial markers but 90% recovery. If the experiment requires highly purified mitochondria for enzyme localization, which method is preferable?

A.Method X, because purity is more important than maximum recovery for this purpose ✅

B.Method Y, because recovery is always more important than purity

C.Method Y, because lower marker enrichment proves better isolation

D.Both are equivalent because purity and recovery cannot be compared

💡 Difficulty: hard | ✅ Correct: A

📖 Explanation: The experimental objective determines the preferred trade-off. When enzyme localization requires a highly enriched mitochondrial fraction, Method X is preferable because its 85% marker enrichment substantially reduces contamination, even though it sacrifices some total recovery.

Q7. A student claims, "If two organelles are the same size, differential centrifugation cannot separate them under any circumstances." Which evaluation is most accurate?

A.Correct, because sedimentation depends only on size

B.Incorrect, because mass, shape, density, and experimental conditions can also influence sedimentation behavior ✅

C.Correct, because centrifugation separates only soluble molecules

D.Incorrect, because differential centrifugation separates particles exclusively according to buoyant density

💡 Difficulty: easy | ✅ Correct: B

📖 Explanation: The claim is too absolute. Sedimentation depends on several physical properties, including particle size, mass, shape, density, and the centrifugal conditions. Similar size does not guarantee identical sedimentation, although overlapping properties can make separation difficult.

Q8. A sample is subjected to isopycnic centrifugation in a density gradient. After equilibrium, two organelles form distinct bands, with organelle A located at a greater density than organelle B. Which conclusion is most justified?

A.Organelle A must be larger than organelle B

B.Organelle A has a higher buoyant density than organelle B under the gradient conditions ✅

C.Organelle B must have sedimented faster throughout the entire experiment

D.Organelle A necessarily contains more DNA than organelle B

💡 Difficulty: easy | ✅ Correct: B

📖 Explanation: At equilibrium in isopycnic centrifugation, particles distribute according to buoyant density within the gradient. A band positioned in a denser region indicates a higher buoyant density, but it does not by itself establish differences in size, DNA content, or sedimentation history.

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