Centrifugation and Molecular Techniques Quiz

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Questions and Answers

Which type of centrifuge is specifically used to separate cells based on their size?

Elutriation Centrifuge (correct)

Laboratory Centrifuge

Benchtop Centrifuge

Ultracentrifuge

What is the primary safety consideration when using an ultracentrifuge?

Avoiding excessive sample volume

Maintaining rotor balance

Using only certified rotors

All of the above (correct)

What is the effect of increasing the density of the medium on sedimentation speed?

The effect cannot be predicted without knowing the density of the particle

Increases sedimentation speed

Decreases sedimentation speed (correct)

Has no effect on sedimentation speed

According to the Svedberg Equation, which factor has the most significant influence on sedimentation speed when the density of the medium is lower than the density of the particles, and the viscosity is low?

Size of the particle (C) Signup and view all the answers

Which of the following is NOT a common application of centrifugation?

Analysis of chemical composition of a sample (C) Signup and view all the answers

What technique or tool is mentioned as widely used for drug discovery?

Microarray (D) Signup and view all the answers

What is a key advantage of Real Time PCR (RT-PCR) mentioned in the content?

Offers high sensitivity for gene expression studies (C) Signup and view all the answers

What is a primary application of FRET-Imaging in the context of the content?

Analyzing protein interactions within cells (A) Signup and view all the answers

What is a key characteriistic of Microarrays that distinguishes them from other techniques?

They can analyze thousands of genes simultaneously (D) Signup and view all the answers

What is a main feature of FRET that distinguishes it from standard fluorescence?

FRET allows for the detection of two signals simultaneously (C) Signup and view all the answers

Which of the following is NOT a common application of chromatography?

Measuring the viscosity of different liquids (C) Signup and view all the answers

What is the role of the stationary phase in chromatography?

To selectively retain different components of the sample (A) Signup and view all the answers

Which of the following is NOT a typical type of chromatography?

Electrophoresis chromatography (B) Signup and view all the answers

What is a major advantage of using Gas Chromatography (GC) for analysis?

It is a highly sensitive technique for detecting and quantifying volatile compounds (A) Signup and view all the answers

What is the function of the carrier gas in Gas Chromatography (GC)?

To provide a constant flow for the mobile phase and transport sample components through the system (D) Signup and view all the answers

What is the primary function of the capillary column in Gas Chromatography?

To provide a surface for the stationary phase (C) Signup and view all the answers

What type of substance is typically analyzed using Gas Chromatography?

Small, volatile compounds such as gases and organic molecules (B) Signup and view all the answers

Which of the following is NOT a common type of detector used in Gas Chromatography?

UV-Vis spectrophotometer (D) Signup and view all the answers

How does Gas Chromatography differ from classical methods of separation, such as precipitation, distillation, and extraction?

It is primarily used to separate and identify components in complex mixtures, unlike classical methods (A) Signup and view all the answers

What is the primary purpose of using a temperature-controlled column in Gas Chromatography?

To optimize the separation of the sample components based on their volatility (B) Signup and view all the answers

Which of the following techniques is NOT commonly used for imaging fast cellular events?

Electron Microscopy (D) Signup and view all the answers

What is the primary advantage of using ratiometric measurement in Calcium Imaging?

It provides an internal control for fluorescence intensity variations. (D) Signup and view all the answers

Which of the following is NOT a characteristic of TRF (Time-Resolved Fluorescence)?

Exclusively used for imaging moving molecules in FRAP experiments. (D) Signup and view all the answers

Which of the following is a common application of TRF-FRET assays?

Identifying specific protein-protein interactions. (C) Signup and view all the answers

What is the primary source of light in bioluminescence?

Chemical reactions within living organisms. (B) Signup and view all the answers

Which of the following is NOT a characteristic of Firefly Luciferase?

It is highly specific for detecting DNA sequences. (D) Signup and view all the answers

Confocal microscopy offers a significant advantage over conventional light microscopy in which aspect?

Enhanced resolution for visualizing thick samples. (C) Signup and view all the answers

What is the fundamental principle behind FRAP (Fluorescence Recovery After Photobleaching)?

Measuring the rate of diffusion of fluorescent molecules. (C) Signup and view all the answers

Which of the following methods for protein quantitation is typically performed at a wavelength of 595 nm?

Bradford (C) Signup and view all the answers

What is the typical wavelength used for measuring the absorbance of NADH cofactor in enzyme kinetic reactions?

340nm (D) Signup and view all the answers

Which of the following methods for protein quantitation is considered to be the most sensitive?

BCA (A) Signup and view all the answers

Which of the following is NOT a characteristic of absorbance at 280nm for protein quantitation?

It is unaffected by the presence of other substances that absorb at 280nm. (A) Signup and view all the answers

In the context of ELISA, what is the primary application of microplate-based photometers?

Protein Quantification (D) Signup and view all the answers

Which of the following statements about the absorbance at 205 nm for protein quantitation is TRUE?

It is not dependent on the protein's amino acid composition. (A) Signup and view all the answers

In dual wavelength measurement, what is the purpose of the second measurement?

To compensate for unspecific signals. (A) Signup and view all the answers

Which of the following is NOT a typical wavelength used as a reference wavelength for ELISA?

405 nm (C) Signup and view all the answers

What is the phenomenon called when a fluorophore emits light at a longer wavelength than the excitation wavelength?

Stokes Shift (D) Signup and view all the answers

What is the primary cause of photobleaching?

Fluorophore degradation due to prolonged exposure to high-energy light sources. (D) Signup and view all the answers

Which of the following is NOT a cause of fluorescence quenching?

Excitation of a fluorophore with light of a specific wavelength. (B) Signup and view all the answers

Why are cyanine dyes considered more resistant to photobleaching?

They have a more stable excited state, reducing their susceptibility to degradation. (A) Signup and view all the answers

What is the main purpose of optical filters in fluorescence detection devices?

To block unwanted light from reaching the detector, separating excitation and emission. (C) Signup and view all the answers

What is the key advantage of using multiplexing in fluorescence detection?

It allows the measurement of multiple fluorophores simultaneously, increasing data throughput. (A) Signup and view all the answers

What is a key feature of a microplate reader for fluorescence detection?

It allows for precise temperature control to optimize fluorescence measurements. (C) Signup and view all the answers

What is a direct consequence of a fluorophore having a shorter lifetime in its excited state?

Higher chance of photobleaching. (A) Signup and view all the answers

Study Notes

Instrumental Analytics (Part 1) - Centrifugation

Centrifugation is a widespread technique used in laboratories across industry and academia for various applications.
Different types of centrifuges exist, including benchtop centrifuges (for low volumes and low speeds), big laboratory centrifuges (for higher volumes), and ultracentrifuges (for high g-forces and separating subcellular components), and elutriation centrifuges (for cell separation based on size).
Ultracentrifuges use vacuum and high rotation speeds (up to 100,000 rpm) to create high g-forces for separating subcellular components.
Centrifugation is a widely used method to separate homogenates (cell mixture) into different fractions.
Fixed-angle rotors are suitable for separating large volumes, while swinging-arm rotors allow greater flexibility during separation.
Safety considerations with ultracentrifuges are crucial due to the high rotational speeds and forces possible, risk of unbalance, and potential for causing catastrophic failure, requiring preventative maintenance and strict adherence to safety protocols.
The proper operation of centrifuge chambers and rotors is essential to ensure the safety and effectiveness of the process.

Instrumental Analytics (Part 1) - Overview

The lecture covers centrifugation, spectroscopy (UV/Vis, fluorescence), flow cytometry, and mass spectroscopy.
The main focus of the lecture is on the application of these instruments rather than just the instruments themselves.
The topics covered include measurements (kinetic and endpoint), various forms of spectroscopy, and a range of applications.

Instrumental Analytics (Part 1) - Flow Cytometry

Flow cytometry is a technique used to analyze cells and other particles based on their physical and biological properties.
It involves passing cells through a small aperture (laser beam) and measuring their characteristics, such as size, complexity, and fluorescence.
Flow cytometry can be used to identify cells that are infected or otherwise dysfunctional.
It can detect a wide range of cells in complex mixtures including determining cell viability and different subtypes (ratios).
Flow cytometry provides a quick way to analyze large numbers of cells in a highly controlled and systematic fashion.

Instrumental Analytics (Part 2) - Spectroscopy

Optical spectroscopy is a technique that uses light (such as UV/Vis, IR, and Fluorescence) to analyse substances.
Spectroscopy can be used to determine the structure, concentration, and other properties of substances.
Spectroscopy is important in biochemical disciplines for structural characterization, concentration measurement, and determining bound/unbound states.

Instrumental Analytics (Part 2) - Special Topics

Photometry is the measurement of light absorbance.
Different coloured liquids absorb different wavelengths of light.
Beer Lambert Law describes the relationship between absorbance and concentration of an absorbing substance.
The unit of absorbance is often Optical Density (OD).
Absorbance is related to transmission (T) by the equation A=-logT where T= I/I₀.

Instrumental Analytics (Part 2) - Photometric Devices

Different types of devices are used for photometry (such as filters and monochromators)
The choice of detector (single photodiode, diode array) depends on sensitivity and range requirements.
A variety of different instruments and sensors are available, and can be used, for example with cuvettes or microplates.
Applications for UV/Vis-Spectroscopy for quantifying proteins, depending on the methodology used.

Instrumental Analytics (Part 2) - Applications for Centrifugation

Centrifugation techniques are used to separate cells, proteins, and nucleic acids.
Methods such as differential centrifugation, zonal centrifugation, and density gradient centrifugation are used to investigate and isolate the elements required to determine these molecules and substances.

Instrumental Analytics (Part 3) - Fluorescence Spectroscopy

Fluorescence spectroscopy is the study of materials that emit light after exposure to another source of light.
Techniques for this include measuring fluorescence intensity, time-resolved fluorescence, and fluorescence polarization.
Fluorescence is used as a tool to examine and quantify biological molecules.

Instrumental Analytics (Part 3) - Fluorescence Applications

High throughput screening (HTS) assays, often use fluorescence-based techniques to locate small and large molecules.
Gene expression analysis is carried out through the use of Fluorescence with technologies such as Microarrays.
Flow cytometry is used as a standard technique, using fluorescent labelled antibodies to measure and determine cell and molecular types and characteristics within complex mixtures, such as infected vs uninfected cell populations and ratios.
Protein and Nucleic Acid quantitation methods using fluorescence-based technologies are performed to quantify protein, peptides, and other specific small molecules.
Fluorescence assays are increasingly important in the study of the function of large and small molecules and their interactions within and between cells.
Fluorescence processes and measurement methodologies, such as Photobleaching and Fluorescence Quenching are frequently utilised in fluorescence assays of biological materials.

Instrumental Analytics (Part 3) - Luminescence Based Spectroscopy

Chemiluminescence and bioluminescence are forms of light emission from chemical reactions and biological systems.
Applications include specific assays for determining the quantity of molecules and biological functions.
Luciferase assays and aequorin assays, often used for determining, for example, the quantity of cellular calcium levels.

Instrumental Analytics (Part 3) - Chromatography

Chromatography is a technique used to separate components of a mixture based on differential partitioning between a mobile and a stationary phase.
Examples include ion exchange chromatography (IEX) for separating molecules based on their charges, hydrophobic interaction chromatography (HIC) separating molecules based on their hydrophobic properties, and affinity chromatography for isolating molecules based on their specific interactions with ligands.
Chromatography is used widely for separating and isolating biomolecules, including proteins, peptides and nucleic acids.

Instrumental Analytics (Part 3) - Electrophoresis

Electrophoresis is a technique that separates charged molecules based on their size and charge in an electric field.
The technique is suited for separating and characterising large molecules such as proteins.
Different methodologies such as Zonal and Isoelectric focussing can be used for separating and analyzing biomolecules.
SDS PAGE and continuous SDS PAGE separate molecules based on their size and charge.
Pulsed Field Electrophoresis gives information on very large molecules.
Examples of separation include proteins, DNA fragments of varying sizes, and other biomolecules.

Instrumental Analytics (Part 3) - 2D-Gel Electrophoresis

A technique used in proteomics by combining isoelectric focussing (IEF) with SDS PAGE.
This allows proteins to be separated based on their isoelectric point (pl) in the first dimension and their size (mass) with SDS-PAGE in the second dimension.
This technique provides a powerful method to identify and quantify proteins and determine and investigate their expression in mixtures and systems/organisms under varying conditions.

Instrumental Analytics (Part 3) - Mass Spectrometry

Mass spectrometry (MS) is used for measuring the mass-to-charge ratio of ions.
Techniques such as MALDI and ESI produce ions, which are then separated by the mass-to-charge ratio or flight time (TOF analyzer)
MS is particularly effective and instrumental in the quantification of molecules such as proteins and peptides through identification and characterisation.

Instrumental Analytics (Part 3) - AK Characterization

This section details the methods used to characterize biomolecules, including primary and secondary structure analysis, higher-order structure analyses, glycosphingolipid, and glycosylation analysis.
Specific methods like ESI-MS, LC-MS, dichroism (CD)), Fourier transform infrared (FT-IR), HDX MS, X-ray diffraction (XRD), and fluorescence spectroscopy provide different levels of detailed information on biophysical properties of the molecules.
These methods are instrumental in determining or investigating the interactions between molecules, and are used to improve understanding of their function.

Instrumental Analytics (Part 3) - Quantification Methods

Absolute methods for quantification require a standard curve
Relative methods for quantification, for example, in gene expression analysis, typically normalize a target gene's expression to a reference gene to determine how significantly different samples are in their composition and gene expressions.

Instrumental Analytics (Part 3) - Real-Time PCR (qPCR)

Real-time PCR, often used to quantify nucleic acid and determining copy numbers.
This method allows for measuring the amplification product in real-time, using fluorogenic probes (e.g., TaqMan or SYBR Green).
Quantitative data, including important data such as Ct values, are obtained.
This method has become important and widespread due to its ability to measure several targets simultaneously (multiplexing).

Instrumental Analytics (Part 3) - Additional Topics

The various instruments and techniques involved, often described with diagrams and models.

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Description

Test your knowledge on centrifugation techniques, including ultracentrifuge applications, sedimentation principles, and cutting-edge methods like Real Time PCR and FRET-Imaging. This quiz covers fundamental safety considerations and characteristics of various laboratory tools used in molecular biology and drug discovery.