Cell Biology: Subcellular Fractionation Basics
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Questions and Answers

What is the primary purpose of subcellular fractionation?

  • To destroy all organelles in the cell
  • To combine organelles from different cells
  • To isolate organelles for biochemical studies (correct)
  • To create new organelles in the cell

Which solution is used to suspend cells prior to disruption of the plasma membrane?

  • Hypertonic solution
  • Hypotonic solution
  • Distilled water
  • Isotonic solution (correct)

What happens to cells placed in a hypotonic solution?

  • They burst immediately
  • They swell due to water influx (correct)
  • They remain unchanged in size
  • They shrink due to water loss

What is the salt concentration of the isotonic solution used for cell suspension?

<p>150 mM (A)</p> Signup and view all the answers

Which of the following organelles is NOT mentioned as part of the subcellular fractionation process?

<p>Chloroplasts (D)</p> Signup and view all the answers

What is the effect of water movement when a cell is in a hypertonic solution?

<p>Water flows out of the cell, causing shrinking (D)</p> Signup and view all the answers

The initial step in subcellular fractionation involves which of the following?

<p>Disruption of the plasma membrane (C)</p> Signup and view all the answers

Which property is essential for creating an isotonic solution for cell studies?

<p>Equal water concentration to that of cell interior (D)</p> Signup and view all the answers

What is the primary effect on cells placed in a hypertonic solution?

<p>The cells shrink as water flows out. (A)</p> Signup and view all the answers

Which concentration defines an isotonic solution compared to the cell interior?

<p>Exactly 150 mM (B)</p> Signup and view all the answers

What is the initial step in subcellular fractionation?

<p>Disruption of the plasma membrane. (D)</p> Signup and view all the answers

Why is it important to use an isotonic solution during cell disruption procedures?

<p>To maintain the normal structure and function of organelles. (B)</p> Signup and view all the answers

What happens when water moves out of cells placed in a hypertonic solution?

<p>The cells and organelles shrink. (C)</p> Signup and view all the answers

Which method is NOT typically used for disrupting cells in subcellular fractionation?

<p>Freezing (A)</p> Signup and view all the answers

What type of vesicles are formed from the endoplasmic reticulum during cell disruption?

<p>Microsomes (A)</p> Signup and view all the answers

What is the purpose of western blotting?

<p>To detect a specific protein in a complex mixture. (D)</p> Signup and view all the answers

Which organelle would be assessed for acid phosphatase activity?

<p>Lysosomes (A)</p> Signup and view all the answers

How does SDS-PAGE affect protein migration?

<p>It results in similar charge density for all proteins. (B)</p> Signup and view all the answers

What is the role of a reducing agent in SDS-PAGE?

<p>To break disulfide bonds between proteins. (B)</p> Signup and view all the answers

Which of the following organelles is likely to be isolated in Fraction #1 based on density?

<p>Peroxisomes (C)</p> Signup and view all the answers

What technique can be used alongside enzymatic assays to assess organelle purity?

<p>SDS-PAGE (C)</p> Signup and view all the answers

What kind of gel is used in western blotting for protein separation?

<p>Polyacrylamide gel (A)</p> Signup and view all the answers

Which of the following correctly describes the migration of proteins in native gel electrophoresis?

<p>Proteins retain their complex structures. (A)</p> Signup and view all the answers

What is the primary benefit of using FRET in cellular studies?

<p>It examines protein-protein interactions. (C)</p> Signup and view all the answers

Which wavelength of light is used to excite CFP in the FRET process?

<p>440 nm (C)</p> Signup and view all the answers

What occurs when proteins Y and Z interact in the context of FRET?

<p>Energy from CFP is transferred to YFP, which emits at 530 nm. (C)</p> Signup and view all the answers

In RNA interference, what is the role of the enzyme Dicer?

<p>It cleaves double-stranded RNA into small fragments. (B)</p> Signup and view all the answers

How are small interfering RNAs (siRNA) generated for RNA interference?

<p>They are generated from DNA plasmids after transfection. (D)</p> Signup and view all the answers

What is the distance within which two proteins must be for FRET to occur?

<p>5 nm (C)</p> Signup and view all the answers

What is the end result of successful RNA interference on a target protein?

<p>The protein is degraded or its expression is reduced. (C)</p> Signup and view all the answers

What type of RNA is involved in RNA interference and specifically targets mRNA?

<p>Small interfering RNA (siRNA) (D)</p> Signup and view all the answers

What is the main purpose of adding an excess of soluble ligand during the elution of the specific protein in affinity chromatography?

<p>To disrupt the interaction between the protein and the ligand (A)</p> Signup and view all the answers

In immunoprecipitation, what indicates that a protein interacts with the target protein?

<p>Detection of other proteins in the pellet (D)</p> Signup and view all the answers

Which organelles are primarily left intact during the cell disruption process?

<p>Nuclei, mitochondria, Golgi, chloroplasts, lysosomes, and peroxisomes (C)</p> Signup and view all the answers

What is the role of the transcriptional activation domain in the yeast two-hybrid system?

<p>To activate the expression of a reporter gene (C)</p> Signup and view all the answers

What happens if the two hybrid proteins do not interact in the yeast two-hybrid system?

<p>Transcription of lacZ will not occur (A)</p> Signup and view all the answers

What term is used to describe the suspension of broken cells after disruption?

<p>Homogenate, extract, or lysate (A)</p> Signup and view all the answers

What type of ligands can be used in affinity chromatography?

<p>Metal ions and proteins (D)</p> Signup and view all the answers

During differential centrifugation, which organelles are likely to sediment first due to their size and density?

<p>Nucleus and mitochondria (D)</p> Signup and view all the answers

How can co-immunoprecipitating proteins be detected after an immunoprecipitation experiment?

<p>Through mass spectrometry or Western analysis (C)</p> Signup and view all the answers

What is the role of the ultracentrifuge in subcellular fractionation?

<p>To apply high centrifugal force for sedimentation (A)</p> Signup and view all the answers

Which organelles form a pellet at a slower rate during differential centrifugation?

<p>Plasma membrane vesicles (A)</p> Signup and view all the answers

What characterizes the specific protein when passing through an affinity chromatography column?

<p>It is attracted to the ligand and retained. (B)</p> Signup and view all the answers

Which of the following components is essential for reconstitution of a transcription factor in the yeast two-hybrid system?

<p>The bait and fish proteins linking to specific domains (A)</p> Signup and view all the answers

How does the size and density of organelles affect their sedimentation in the centrifugation process?

<p>Heavier organelles sediment more quickly than lighter ones (A)</p> Signup and view all the answers

What is the primary benefit of conducting cell disruption in an isotonic solution on ice?

<p>To avoid damaging the organelles (D)</p> Signup and view all the answers

Which of the following organelles are sedimented last during differential centrifugation?

<p>Microsomes and plasma membrane vesicles (D)</p> Signup and view all the answers

Flashcards

Subcellular Fractionation

A process used to separate different organelles from eukaryotic cells.

Isotonic Solution

A solution that has the same salt concentration as the inside of a cell.

Hypotonic Solution

A solution that has a lower salt concentration than the inside of a cell.

Hypertonic Solution

A solution that has a higher salt concentration than the inside of a cell.

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Osmosis

The process of water moving across a membrane from an area of high water concentration to an area of low water concentration.

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Cell Swelling

The swelling of cells when placed in a hypotonic solution due to water flowing into the cell.

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Cell Shrinking

The shrinking of cells when placed in a hypertonic solution due to water flowing out of the cell.

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Cell Disruption

The process of disrupting the plasma membrane and cell wall (if present) of cells to release the organelles.

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Differential Centrifugation

A technique using centrifugal force to separate cellular components based on size and density.

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Homogenate (lysate, extract)

A suspension of broken cells created by disruption. It contains the released organelles and other cellular components.

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Pellet

The pellet formed at the bottom of a centrifuge tube during differential centrifugation, containing organelles or cellular components of different sizes and densities.

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What is an isotonic solution?

A solution that has the same salt concentration as the inside of a cell, leading to no net movement of water in and out of cells. Cells do not swell or shrink in such solutions.

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What is a hypertonic solution?

A solution that has a higher salt concentration than the inside of a cell. Water moves out of cells until the ion concentration inside and outside becomes equal. This leads to cell shrinking.

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What is a hypotonic solution?

A solution that has a lower salt concentration than the inside of a cell. Water moves into cells until the ion concentration inside and outside becomes equal. This leads to cell swelling.

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What is cell disruption?

A process that breaks open cells to release their organelles. It's done under specific conditions to preserve the integrity of the organelles.

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What are microsomes?

Small, closed vesicles formed when the plasma membrane or ER breaks apart during cell disruption.

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What is subcellular fractionation?

The process of separating different organelles using specific techniques, such as centrifugation, to study their individual functions.

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What is sonication?

A technique used to disrupt cells by exposing them to high-frequency sound waves.

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What is a homogenizer?

A device used to physically disrupt cells by forcing them through a narrow space.

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Density gradient centrifugation

Organelles are collected in different fractions based on their densities.

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Assessing organelle purity

The purity of organelle preparations can be assessed by examining their morphology under an electron microscope or by measuring their enzymatic activity.

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Protein electrophoresis

A technique used to separate proteins based on their size and charge, typically using a polyacrylamide gel.

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SDS-PAGE

A type of protein electrophoresis using a detergent that denatures proteins, allowing separation based on size.

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Western Blot

A technique used to detect specific proteins in a sample by using antibodies that bind to the target protein.

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Marker enzymes

Enzymes found within specific organelles that can be used as markers for organelle identification.

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Enzymatic assay

A technique used to measure the activity of an enzyme within a sample.

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Affinity Chromatography

Affinity chromatography separates proteins based on their specific binding affinity to a ligand immobilized on a column. The target protein binds to the ligand, while other proteins flow through. Elution releases the target protein by disrupting the interaction.

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Immunoprecipitation

Immunoprecipitation uses antibodies as ligands in affinity chromatography to isolate a specific antigen (protein) from a cell extract. The antibody binds to its target, and the complex is collected by centrifugation.

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Yeast Two-Hybrid

The yeast two-hybrid system exploits the interaction between two proteins to reconstitute a functional transcription factor, triggering the expression of a reporter gene (e.g., lacZ). This allows detection of protein interactions.

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FRET (Förster Resonance Energy Transfer)

A method for studying protein interactions that relies on the transfer of energy between two fluorescent molecules. If the proteins interact, their fluorescent properties change, indicating an interaction.

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Ligand in Affinity Chromatography

In affinity chromatography, a specific molecule (ligand) is attached to a solid support (e.g., a column). This ligand binds to a specific protein, allowing for its separation from a mixture.

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Elution in Affinity Chromatography

Elution in affinity chromatography involves releasing the bound target protein from the ligand by using a solution that disrupts the interaction. This allows isolation of the purified protein.

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Retention in Affinity Chromatography

In affinity chromatography, the target protein is attracted to the ligand and retained on the column. This difference in binding affinity is what allows separation.

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Centrifugation in Immunoprecipitation

In Immunoprecipitation, the antibody-antigen complex is collected by centrifugation, separating the target protein from the rest of the cellular extract.

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What is FRET (Fluorescence Resonance Energy Transfer)?

A technique that measures the distance between two fluorescent molecules by detecting their energy transfer, used to study interactions between proteins within a cell.

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How does FRET work in protein interactions?

When two proteins labeled with CFP and YFP are close enough (<5nm), excitation of CFP at 440nm causes energy transfer to YFP, resulting in strong emission at 530nm.

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What happens in FRET if proteins do not interact?

If the proteins do not interact, CFP will emit light at 480nm with minimal emission at 530nm because energy transfer to YFP does not occur.

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What is RNA interference (RNAi)?

A cellular process that uses double-stranded RNA to specifically target and degrade mRNA, thereby silencing the expression of a particular gene.

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What are siRNAs?

Small interfering RNAs (siRNAs) are short (~21bp) double-stranded RNA molecules generated from a longer double-stranded RNA by the enzyme Dicer.

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How do siRNAs work in gene silencing?

siRNAs are incorporated into the RNA-induced silencing complex (RISC) where they guide the complex to target mRNA molecules for degradation.

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Why is RNAi important for studying gene function?

RNAi provides a powerful tool for studying gene function by selectively silencing the expression of a specific gene and observing the resulting cellular effects.

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What is the Central Dogma of Molecular Biology?

The central dogma explains the flow of genetic information from DNA to RNA to protein, which is the foundation of molecular biology.

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Study Notes

Cell Biology - Unit 2: How Cells Are Studied - II

  • Subcellular Fractionation: A technique to isolate organelles from eukaryotic cells for biochemical study. This involves separating the cell into its functional organelles.

  • Subcellular Fractionation - Initial Step: Disruption of the plasma membrane and cell wall (if present) using conditions that don't destroy the organelles. The cells are suspended in an isotonic solution with appropriate pH (~7.5) and salt content. Maintaining an isotonic solution is crucial to prevent cell swelling or shrinkage. This process keeps organelles intact.

  • Isotonic Solution: Has a salt concentration (150 mM) equal to that of the cell interior. Water flows equally across the plasma membrane in this solution, so cells maintain their normal size and shape.

  • Hypotonic Solution: Has a lower salt concentration than the cell interior. Water flows into the cells, causing them to swell, potentially leading to lysis of organelles.

  • Hypertonic Solution: Has a higher salt concentration than the cell interior. Water flows out of the cells, causing them to shrink.

  • Methods for Cell Disruption:

  • High-speed Blending: Stirring the cell suspension in a high-speed blender.

  • Sonication: Exposing the cell suspension to high-frequency sound waves.

  • Mechanical Homogenization: Grinding the cells in a mechanical homogenizer.

  • Homogenization Outcomes: Breaks the plasma membrane and the ER membrane into small fragments. Some fragments immediately reseal, forming small vesicles (microsomes), which are derived from the ER. Nuclei and other organelles are present, but not disrupted as much

  • Centrifugation (Differential): Initial step in most subcellular fractionation procedures; increasing speeds fractionate based on size and density. Largest/heaviest organelles pellet first. Different speeds pellet different organelles in order; such as nuclei then mitochondria, then ER fragments.

  • Ultracentrifugation: A more powerful type of centrifugation.

  • Higher speeds (200,000 rpm) creating greater centrifugal force exceeding gravity by ~500,000 times.

  • This separates microsomes and organelles even more thoroughly than differential.

  • Differential Centrifugation Results/Further Purification (equilibrium density-gradient):

  • Nuclei: Sediment first, at low speeds (1000 x g for 10 minutes)

  • Mitochondria, lysosomes, Golgi, peroxisomes: Pellet next at higher g-force (20,000 x g for 20 minutes)

  • Microsomes (ER) and plasma membrane vesicles: Pellet last, at highest g-force (100,000 x g for 1 hour)

  • Cytosol: Remains as the supernatant even after maximum g-force.

  • Equilibrium Density-Gradient Centrifugation: Technique to further purify organelles; layers of sucrose solution with increasing density. Organelles separate based on their density, not size, in this form of centrifugation.

  • Sucrose Density Gradient: Sucrose has varying concentrations to create layers with increasing density for optimal fractionation of cellular components based on density. Organelles sediment to a position where the density matches their own density within the gradient.

  • Organelle Density Values: Numerical values provided for density of different organelles.

  • Western Analysis: A technique to detect a specific protein in a sample. Separates proteins by size in a polyacrylamide gel; transferred to a membrane; probed with a specific antibody; antibodies detected with added color or light for visualization.

  • Protein Separation in Western Analysis: Western blotting is used to detect a specific protein in a sample with many other proteins with the aid of separating proteins on polyacrylamide gel.

  • Western Analysis - Steps:

  • Separate proteins by gel electrophoresis.

  • Transfer the proteins onto a thin membrane.

  • Label the membrane with specific antibodies targeting a protein of interest.

  • Identify the antibodies using a substrate or other method (e.g., colored solution) to indicate where they have attached to the membrane.

  • Chromatography: Multiple techniques for separating proteins from other cellular components:

  • Ion exchange: Separates proteins based on charge.

  • Size exclusion (gel filtration): Separates proteins based on size.

  • Affinity: Separates proteins based on a specific sequence.

  • Protein Interactions:

  • Immunoprecipitation: Detects protein-protein interactions (interaction with antigen).

  • Yeast two-hybrid: Allows for reconstituting a transcription factor by the interacting proteins. Expresses a reporter gene (easily measurable), that shows protein interactions.

  • FRET (Fluorescence Resonance Energy Transfer): Detects protein-protein interactions using fluorescent proteins.

  • Gene Silencing (RNA interference, RNAi): Techniques to remove a specific protein from a cell by removing the mRNA related to the protein. Used to assess the protein function.

  • siRNA (small interfering RNA): RNA molecules capable of silencing a specific mRNA, breaking down the mRNA transcript after being transfected into a cell.

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Biol 266 - Cell Biology Unit 2 PDF

Description

This quiz covers essential concepts related to subcellular fractionation, including the effects of different solutions on cells and the significance of isotonic conditions during cell studies. Test your knowledge on the procedures and principles involved in the separation of cellular components.

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