Cell Biology Chapter 11 & 14 Quiz
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Questions and Answers

What is the principle behind ion-exchange chromatography?

  • It relies on the temperature of the solution.
  • It uses the charge of proteins to separate them. (correct)
  • It separates proteins based on their size.
  • It separates proteins based on their affinity to a specific substrate.

What is a primary advantage of using TIRF microscopy during imaging?

  • It allows for deeper tissue penetration.
  • It minimizes background light by illuminating only a thin layer. (correct)
  • It uses higher-energy light to enhance imaging speed.
  • It can illuminate the entire sample at once.

How does confocal microscopy improve the clarity of images?

  • By employing a pinhole to focus on a specific plane. (correct)
  • By cooling the sample to reduce movement.
  • By using fluorescence from all layers of the sample.
  • By using multiple color filters.

What is a key benefit of using multi-photon microscopy compared to traditional confocal microscopy?

<p>It facilitates deeper imaging with less damage. (D)</p> Signup and view all the answers

What does GFP technology allow researchers like Dr. Smith to do in their experiments?

<p>Track the dynamics of proteins in live cells. (C)</p> Signup and view all the answers

How does multi-photon microscopy reduce damage during imaging?

<p>By utilizing lower-energy infrared light. (C)</p> Signup and view all the answers

What happens to proteins during affinity chromatography?

<p>Target proteins bind to specific molecules in the column. (B)</p> Signup and view all the answers

Which of the following is a common issue Dr. Smith needs to address during his imaging process?

<p>Background noise affecting image clarity. (C)</p> Signup and view all the answers

What is the main advantage of using size-exclusion chromatography in protein isolation?

<p>It separates proteins on the basis of their size. (A)</p> Signup and view all the answers

In FRET analysis, what is indicated by a decrease in fluorescence from one protein and an increase in fluorescence from another?

<p>The proteins are interacting closely. (B)</p> Signup and view all the answers

What conclusion can be drawn if a researcher observes rapid fluorescence recovery after bleaching in FRAP experiments?

<p>The proteins are highly mobile. (C)</p> Signup and view all the answers

How does super-resolution microscopy like STORM improve imaging over conventional microscopy?

<p>It achieves resolutions at the nanometer scale. (D)</p> Signup and view all the answers

What is the purpose of differential centrifugation in protein isolation?

<p>To collect the supernatant containing soluble proteins. (B)</p> Signup and view all the answers

What role does ion-exchange chromatography play in protein purification?

<p>It separates proteins based on their charge. (D)</p> Signup and view all the answers

Which microscopy technique allows for the visualization of specific proteins using a fluorescent tag like GFP?

<p>Confocal microscopy. (C)</p> Signup and view all the answers

What is a key feature of TIRF microscopy compared to traditional optical microscopy?

<p>It only illuminates a thin region of the sample. (B)</p> Signup and view all the answers

How does gel filtration chromatography separate proteins?

<p>By size, using beads with tiny holes (A)</p> Signup and view all the answers

What is a primary benefit of using confocal microscopy?

<p>It only captures light from the focal plane, reducing blur (C)</p> Signup and view all the answers

What distinguishes differential centrifugation from density gradient centrifugation?

<p>Differential centrifugation spins extracts at increasing speeds (B)</p> Signup and view all the answers

Which of the following is true regarding multi-photon imaging?

<p>It allows deeper tissue penetration with less damage (D)</p> Signup and view all the answers

What role does electrophoresis serve in protein analysis?

<p>It visualizes proteins while confirming size and purity (C)</p> Signup and view all the answers

How does TIRF microscopy differentiate from standard fluorescence microscopy?

<p>It excites only the fluorescent molecules close to the surface (A)</p> Signup and view all the answers

What is one of the main applications of GFP technology in research?

<p>For real-time visualization of protein localization (A)</p> Signup and view all the answers

What advantage does density gradient centrifugation offer for separating cell components?

<p>It separates components based on their varying densities (D)</p> Signup and view all the answers

Flashcards

Ion-exchange chromatography

Separates proteins based on their charge.

Affinity chromatography

Separates proteins based on their specific binding to molecules.

Confocal Laser-Scanning Microscopy (CLSM)

Microscopy technique that distinguishes in-focus and out-of-focus light. Reduces background noise.

Pinhole aperture in CLSM

Filters out-of-focus light from the sample in confocal microscopy, improving image clarity.

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TIRF Microscopy

Microscopy technique reducing background noise by targeting a specific layer.

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GFP technology

Attaching GFP gene to protein of interest enabling tracking through fluorescence.

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FRAP technique

Used by Dr.Smith to track protein dynamics over time in living cells.

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Multi-photon microscopy

Microscopy technique that reduces sample damage by using lower-energy light.

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GFP function

GFP (Green Fluorescent Protein) is a jellyfish protein that glows green under certain light. It can be attached to a gene of a protein to allow the cell to produce a combined protein, enabling location, tracking and behavior observation.

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FRET analysis

A technique to study protein interactions. Close protein interaction leads to a change in fluorescence of one protein, where one loses and the other gains fluorescence.

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FRAP experiment

Measures protein mobility by observing fluorescence recovery after bleaching. Fast recovery implies high mobility.

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Super-resolution technique (STORM)

Enhances microscopy resolution, allows for nanometer-scale visualization, overcoming limitations of conventional microscopes.

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Differential Centrifugation

Separates cellular components by size and density, allowing collection of supernatant containing soluble proteins.

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Size-exclusion chromatography

Separates proteins based on size. Larger proteins elute first, smaller proteins are retained longer in the column

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SDS-PAGE

Analyzes protein size after chromatography. Separates proteins by size (mass).

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Confocal Microscopy

A microscopy technique that uses a laser to illuminate a specific focal plane, reducing blur by filtering out-of-focus light.

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Electrophoresis

A technique separating proteins based on their size and charge using an electric field.

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Cell Fractionation

Breaking open cells to release their contents (proteins, organelles) and separate them based on their properties.

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Density Gradient Centrifugation

A method separating components based on their density, where a gradient medium is used.

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Gel Filtration Chromatography

Separates proteins based on size by passing them through a column packed with beads.

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What is the goal of protein purification?

To isolate and purify a specific protein from a complex cellular mixture for further study.

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What does SDS do in protein analysis?

SDS (Sodium dodecyl sulfate) denatures proteins and gives them a uniform negative charge, allowing separation based on size.

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Study Notes

Week 8 (Oct 24): Mitochondria & Chloroplasts – Structure

  • Chapter 11: pages 637-651, 653 (ion channels) - 661
  • Chapter 14: pages 811-817, 861-868
  • Chapter 12: pages 726-735
  • Chapter 14: pages 843-844

Confocal Laser-Scanning Microscopy

  • Confocal laser scanning only allows light from the exact focal plane to be captured
  • This prevents light from areas above or below the focal point, reducing blurry images.
  • Electrophoresis is used to separate proteins based on size and charge
  • SDS protein gives a uniform negative charge, making proteins linear
  • This allows visualization for protein size and purity
  • Confirm correct separation for further studies or applications

Cell Fractionation

  • Cell fractionation breaks open cells to release contents (proteins, organelles) to create a cell extract
  • The cell extract allows separation of cellular components
  • Centrifugation and chromatography are used in protein purification
  • These methods separate proteins by size, charge, or other properties

Differential vs. Density Gradient Centrifugation

Feature Differential Centrifugation Density Gradient Centrifugation
Method Spins extract at increasing speeds Uses a sucrose gradient
Separation Heavier parts settle first (nuclei) Components move at different speeds, forming distinct bands
Basis Looks at the mass of components Looks at the size of components

Column Chromatography

  • Gel filtration: Separates proteins by size. Larger proteins pass through holes faster than smaller ones.
  • Ion exchange: Separates proteins by charge. Proteins with opposite charge are attracted to the column, separating them by changing salt or pH.
  • Affinity: Separates proteins based on binding to specific molecules. The column binds to proteins of interest. Other molecules pass through without binding.

Super-Resolution Techniques

  • STORM: Enhanced imaging capabilities compared to conventional microscopy,
  • Allows scientists to see details at the nanometer scale (smaller than conventional light microscopes)

Protein Isolation Methods

  • Differential centrifugation: Separates cellular components by size and density.
  • Size-exclusion chromatography: Separates proteins based on size. Larger proteins elute first.
  • Ion-exchange chromatography: Separates proteins based on charge.
  • SDS-PAGE: Separates proteins based on size after electrophoresis. SDS gives proteins a uniform negative charge, denaturing them.

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Description

Test your knowledge on mitochondria and chloroplasts as covered in Chapters 11 and 14. This quiz focuses on the structures, functions, and methodologies such as confocal laser-scanning microscopy and cell fractionation. Review your understanding of protein separation techniques and cellular components.

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