Cell Biology: Centrifugation Techniques

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Questions and Answers

Which type of centrifuge is best suited for separating subcellular components at very high speeds?

Benchtop centrifuge

Swing-out rotor centrifuge

Elutriation centrifuge

Ultracentrifuge (correct)

What is the primary safety consideration when using ultracentrifuges?

Calibrating the centrifuge frequently

Using specified rotors and avoiding unbalance (correct)

Choosing the smallest samples possible

Ensuring rotors are cleaned regularly

In the Svedberg equation for sedimentation speed, which factor is most critical when centrifugation occurs in media with lower density and low viscosity?

Size of the particle (d) (correct)

Density of the medium (ρ(m))

Relative centrifugal acceleration (g)

Viscosity of the medium (η)

Which type of centrifuge is best for handling low volumes and operates at low speeds?

Benchtop centrifuge Signup and view all the answers

What is the purpose of using specified sample holders in centrifugation?

To ensure proper distribution of weight and prevent unbalance Signup and view all the answers

What is the consequence of light interacting with materials?

Electrons can be elevated to higher energy levels. Signup and view all the answers

What does the Beer-Lambert law relate?

Absorbance and concentration of the absorbing substance. Signup and view all the answers

Which of the following statements about excited states is true?

Excited states can lead to fluorescence or phosphorescence. Signup and view all the answers

What measurement unit is used for absorbance in photometric measurements?

Optical Density (OD) Signup and view all the answers

Which characteristic of UV light contributes to sunburn?

High energy Signup and view all the answers

What is the primary purpose of differential centrifugation in cell separation?

To pellet different cellular components based on density Signup and view all the answers

In zonal centrifugation, what characteristic of the medium is essential for effective particle separation?

Density gradient with a maximum density greater than the lowest particle density Signup and view all the answers

Which medium is known for its high osmolarity and is primarily used for nucleic acid separation?

Cesium Chloride Signup and view all the answers

What is a key advantage of optical spectroscopy in biochemical analysis?

It has minimal requirements on sample preparations and work load Signup and view all the answers

Which optical spectroscopy technique is used to determine the structure of molecules?

Circular Dichroism (CD) Signup and view all the answers

What is the relationship between transmission (T) and optical density (OD)?

T decreases as OD increases. Signup and view all the answers

At what wavelength is the Bradford assay typically measured?

595nm Signup and view all the answers

How is light intensity after passing through the sample denoted in the equations?

I Signup and view all the answers

Which photometric device classification provides continuous spectrum selection?

Monochromator Signup and view all the answers

Which of the following statements regarding absorption at 280nm is true?

It indicates the presence of tryptophan and tyrosine residues. Signup and view all the answers

What is the main application of microplate-based photometers?

ELISA Signup and view all the answers

What is indicated by a reading of A280 = 1 in terms of protein concentration?

0.5 – 2 mg/ml protein concentration Signup and view all the answers

What type of detector is often found in modern spectrophotometers for enzyme kinetics?

Diode array Signup and view all the answers

What is the purpose of blank measurement in single wavelength assays?

To compensate for light scattering by the cells Signup and view all the answers

What is the typical reference wavelength used in dual wavelength ELISA measurements?

620-650 nm Signup and view all the answers

Which of the following molecules are typically analyzed using Circular Dichroism spectroscopy?

Optically active chiral molecules Signup and view all the answers

What characteristic is measured to quantify Circular Dichroism?

Mean residue ellipticity Signup and view all the answers

Which secondary structure components of proteins can be analyzed with CD spectroscopy?

Helices, Sheets, Turns, and Coils Signup and view all the answers

How does Circular Dichroism spectroscopy differentiate between chiral molecules?

By analyzing the difference in absorption of circularly polarized light Signup and view all the answers

In dual wavelength measurement, what happens at the reference wavelength?

It is subtracted from the target wavelength absorbance Signup and view all the answers

During the analysis of protein secondary structures, what wavelength range is scanned using CD spectroscopy?

190 nm to 240 nm Signup and view all the answers

Study Notes

Instrumental Analytics

The course covers instrumental analysis, with a focus on applications
The instructor is Harald Hundsberger at the University of Applied Sciences, Krems
The semester is Wintersemester 23/24

INSA Overview

Techniques like centrifugation, UV/Vis spectroscopy, fluorescence spectroscopy, chromatografy, electrophoresis, and mass spectroscopy are covered
Applications include kinetic and endpoint measurements, RT-PCR, HTS, sequencing, CE, laser scanning microscopy, and more
Principles of IEX, FPLC, HPLC, electrophoresis/CE, 2D electrophoresis, and mass spectroscopy are addressed

Flow Cytometry

The lecture focuses on applications of flow cytometry, despite covering the instruments
Measurements include forward and side scatter
Forward scatter measures cell size, and side scatter measures internal complexity
A relevant online resource is provided: https://oncohemakey.com/principles-of-flow-cytometry/

Part I Centrifugation

A technique widely used in labs and academia
Various centrifuge types are used for diverse applications
- Benchtop centrifuges: low volumes, low speeds
- Larger centrifuges: higher volumes (liters)
- Ultracentrifuges: high G-forces, vacuum applied (100,000 rpm) for subcellular component separation
- Elutriation centrifuges: separation by size

Centrifugation - Types of Centrifuges

Benchtop centrifuges are used for low-volume, low-speed applications.
Larger centrifuges are used for higher sample volumes (up to several liters).
Ultracentrifuges use vacuum and high rotational speeds (100,000 rpm) to separate subcellular components due to high G-forces.
Elutriation centrifuges separate cells by size.

THE CENTRIFUGE

Centrifugation separates a homogenate into components
The technique uses rotors (fixed-angle or swinging-arm) with varying capacities and pellet evenness
Centrifuge speed and time are crucial for successful separation
Refrigerated and evacuated chambers are essential for high speeds to avoid heating during centrifugation and for preventing rotor imbalance

Safety Considerations UC

Using specified rotors and sample holders is crucial for safety
Sample weight and balance are critical, often requiring sub-milligram accuracy
Equipment has specified lifetimes

Classification on Rotor Type

Fixed-angle rotors hold larger quantities than swinging-arm rotors
With fixed-angle rotors pellets form less evenly

Centrifugation - Basics

Svedberg equation (V = d² (Pp-Pm)g / 18η) relates sedimentation speed (V) to particle size (d), particle density (Pp), medium density (Pm), medium viscosity (η), and relative centrifugal acceleration (g)
Particle size is crucial for sedimentation, along with density and viscosity

Density-Gradient Centrifugation

Gradients (often sucrose) separate particles based on density
The highest density of the medium needs to be higher than the separated particles

Media for density Gradients

Cesium chloride (CsCl): high osmolarity, used for nucleic acids separation (advantage of low viscosity, disadvantage of high osmolarity)
Sucrose: non-ionic, no interactions with biological material (advantage), low resolution (disadvantage)
Percoll: good osmolarity capabilities for whole organelle preparations

Applications for Centrifugation

Cell harvesting (e.g., yeast, bacteria from fermentations)
Protein purification
Desalting and concentrating proteins (Amicon tubes)
Nucleic acid pellet (Plasmid prep, RNA prep)
Spin columns (Silica based)
Subcellular fractionation
Cell separation (elutriation)

Part II - Spectroscopy Basics

Little sample prep needed
Well-defined laboratory equipment is utilized
UV/Vis, CD, fluorescence

Optical Spectroscopy

Requires minimal sample preparation
Standardization is key
Applications in biological disciplines include structure determination (CD), concentration measurement (UV/Vis, Fluorescence), bound/unbound state analysis (Fluorescence Polarization), reaction kinetics (Fluorescence, Absorbance), and substance analysis (Absorbance, IR-Spectroscopy).

What is Light?

Light is an electromagnetic wave with oscillating electric and magnetic fields
Light exhibits wave-particle duality

Polarized Light

Light can vibrate in various directions
Plane-polarized light vibrates in a single direction.
Filter, vibration direction.
Circularly polarized light vibrates in a circular pattern
Elliptical polarization vibrates in an ellipse.

Electro Magnetic Spectrum

The electromagnetic spectrum spans a broad range of wavelengths at varying frequencies and energies
UV light can cause sunburn

Interaction of Light and Material/Sample

Photons interact with material by raising electrons to higher energy levels
Transitions between energy levels are extremely fast
Light can be absorbed without subsequent radiation after the initial energy exchange, resulting in excitation
Excited states exist for finite times and are unstable

Part III - UV/Vis/NIR Spectroscopy

UV/Vis/NIR spectroscopy deals with measurement of specific absorbance of light at particular wavelengths

Basics of Photometry

Photometry measures specific absorbance (OD) of light
Colored liquids absorb specific wavelengths
Beer-Lambert law relates absorbance to concentration.

Photometric Measurements

Concentration determination through spectrophotrometric analysis
Path length measurements require standardized 1cm cuvettes
Standard curves aid in determining molar extinction coefficients when not known.

Photometric Measurements (continued)

Absorbance measures light intensity after passing through a sample, using optical density (OD) values
Optical density (OD): measures absorbance using a logarithmic scale (A = -log T) and can be derived from the transmission of light through the sample.

Relationship Transmission and OD

Transmission (T) and absorbance (OD) are inversely related
Different percentages of transmission equate to different OD readings

Classification of Photometric Devices

Different photometers include single-beam, dual-beam instruments, with varied monochromators and detectors
Monochromators select wavelengths, and single photodiodes/diode arrays measure transmitted light

Specifications

Provided technical specifications for DU 800 performance

Applications UV/Vis Spectroscopy

Protein quantification (Biuret, Bradford, Lowry, BCA methods)
DNA quantification
Enzyme kinetics measurement at 340nm, using NADH cofactor
- Proteases: use available colorimetric substrates

Applications UV/Vis Spectroscopy (continued)

Cell viability measurements using tetrazolium salts (XTT)
ELISA (Enzyme-Linked Immunosorbent Assay)

Measurement Modes (Single Wavelength)

Measures absorbance or optical density at a single wavelength
Necessary background or blank measurement to account for light scattering

Measurement Modes (Dual Wavelength)

Measures absorbance (optical density) at two wavelengths simultaneously
Used for compensation in assays like ELISA
Employing substrates for alkaline phosphatase (AP) including pNPP (405 nm) and aminoantipyrene/phenyl phosphate (492 nm), with reference wavelengths (620-650nm).

Part V - Circular Dichroism Spectroscopy

CD determines absorbance differences of left and right circularly polarized light when light is passed through a chiral molecule
CD analysis typically involves scans from 190-240nm to yield secondary structure information

Circular Dichroism Spectroscopy (continued)

Chiral molecules exhibit different absorption of left and right circularly polarized light
CD provides spectral information about the secondary structure of protein structures (helices, sheets, turns, coils)
CD measurements expressed in ellipticity

Biosimilar Analytics, Rituximab

UV/Vis spectroscopy used in biosimilar analytics to assess properties of Rituximab (a monoclonal antibody)

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Description

Test your knowledge on centrifugation techniques used in cell biology, including ultracentrifuges, differential centrifugation, and the Beer-Lambert law. This quiz covers safety considerations, measurement units for absorbance, and factors influencing sedimentation speed. Assess your understanding of these essential laboratory methods!