lECTURE 14 (Research Techniques) Flashcards
What is Sedimentation?
The motion of particles in a fluid under the application of a force
Example: Snow particles in a snow globe slowly descend in water under the force of gravity
What does the sedimentation rate of a particle/molecule depend on?
- Force
- Density of the fluid
- Size & density (or concentration) of particles in fluid
What does applying a force stronger than gravity do?
- Increase the sedimentation rate
- Magnify differences in sedimentation behaviour between different molecules
[makes ultracentrifugation a convenient technique for separating molecules of different sizes]
What is Centrifugation?
A technique that applies a centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed
Explanation: More dense components of the mixture migrate away from the axis of the centrifuge, while the less dense components of the mixture migrate towards the axis
What is a Centrifuge?
A device driven by an electric motor that puts an object in a rotational movement around a fixed axis
What is the correlation between the size and density of a particle and the rate that the particle separates from a heterogenous mixture?
The larger the size and the larger the density of the particles, the faster they separate from the mixture -> a larger gravitational force accelerates the separation of the particles
What are the properties of the rate of centrifugation?
- Specified by the angular velocity expressed as REVOLUTIONS PER MINUTE (RPM) or acceleration expressed as “g”
- Conversion factor between RPM and “g” depends on the radius of the centrifuge rotor
What is the particles’ settling velocity in centrifugation dependent on?
- Size and shape
- Centrifugal acceleration
- Volume fraction of solids present
- Density difference between the particle and the liquid
- Viscosity
What is the formula for calculating revolutions per minute (RPM)?
RPM = square root g/r
g - respective force of the centrifuge
r - radius from the center of the rotor to a point in the sample
Explanation: As suspension is rotated at a certain speed/RPM, centrifugal force allows particles to travel radiant away from the rotation axis
How does gel increases the differences in sedimentation rates between molecules of different sizes within the gel?
The molecular structure of the gel acts to obstruct the movement of molecules within the gel -> Larger molecules are more easily obstructed than small molecules
What is Sedimentation in Electrophoresis affected by?
- Density of gel
- Size and shape of molecules
- Charge on the molecules
What is Electrophoresis?
A laboratory technique used to separate DNA, RNA or protein molecules based on their size and electrical charge and an electric current is used to move molecules to be separated through a gel
What is the Mechanism of Electrophoresis?
- Electric charges attract their objects to their respective poles
- Since DNA fragments are negatively charged, they will move towards the positive pole of the electrophoresis chamber
What are the properties of Agarose gel?
- Used for electrophoresis of DNA
- Large pore structure allowing larger molecules to move easily
- Not suitable for sequencing smaller molecules
What has a clearer solution, PAGE or Agarose gel?
PAGE (Polyacrylamide gel electrophoresis)
Explanation: more suitable for quantitative analysis
What are the properties of 2D Electrophoresis?
- Used to resolve complex mixtures of thousands of proteins
- separates proteins in 2 steps: Isoelectric Focusing (IEF) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
- Information about IEF and molecular weights can be obtained
What is the difference between IEF and SDS-PAGE?
IEF = separates proteins according to isoelectric points
SDS-PAGE = separates proteins based on the molecular weights
What are the steps for successful 2D-Electrophoresis?
1) Sample preparation = process will result in the complete solubilisation, disaggregation, denaturation and reduction of proteins in the sample
2) First dimension: Isoelectric focusing (IEF) = +ve net charge protein towards cathode, -ve net charge protein towards anode -> both becoming less charged towards reaching its Isoelectric points
3) Second dimension: SDS-PAGE = preparation of gel, equilibrium of the immobilised pH gradient (IPG) strips in SDS buffer, placing the equilibrated IPG strip on the SDS gel and handling the electrophoresis
4) Visualisation of results: staining = Coomassie Blue staining + Silver staining
Describe Silver staining
- Sensitive + non-radioactive
- Amino acid side chains bind to silver ions (sulfhydryl + carboxyl groups of proteins) -> reduction to free metallic silver -> protein bands are sports where reduction occurs
- Suitable for low protein levels because of sensitivity
Describe Coomassie Blue staining
- More quantitative than silver staining
- Suitable to detect protein bands containing 0.2 μg or more proteins
- Dye binds to proteins to form a protein-dye complex through Van Der Waals interactions
- Two types of dyes: R250 and G-250
What are IPG strips?
Immobilised pH gradient (IPG) strips facilitate Isoelectric focusing -> Each sample protein applied to an IPG strip will migrate to its isoelectric point (the point at which its net charge is 0)
What does Spectroscopy involve?
Sending some form of electromagnetic radiation into a sample and measuring various properties of the electromagnetic radiation that emerges from the sample
Examples: Intensity, Direction of emitted radiation & Polarisation
What is the equation to calculate frequency?
f = v/λ
f - frequency
v - speed of light
λ - wavelength
What is the equation to calculate Energy?
E = hf
E - Energy
h - Planck’s constant
f - frequency
What happens in Absorption spectroscopy?
A light of specific wavelength is shone through a sample and you measure the intensity of light that comes out the other side -> measures absorbance
What does the absorbance of a given sample depend on?
- Intrinsic ability of molecules in solution to absorb light
- Concentration of molecules in solution
- Path length of light as it passes through the sample
What is an Absorption spectrum?
A graph of the absorbance versus the wavelength/frequency of light
What are the different types of Spectra?
- Emission spectrum
- Absorption spectrum
Describe Emission spectrum
When atoms or molecules are subjected to intense heat or electric discharge, they absorb energy and get excited -> Since lifetime of electrons is short, they return to lower state and energy is absorbed as light energy -> When passes through a prism, it gives rise to an emission spectrum
Describe Absorption spectrum
When an electromagnetic radiation is passed through a solution, some is absorbed, some is reflected and remainder is transmitted -> Transmitted light is passed through a prism and absorption spectra is obtained
What are the properties of UV/Visible Spectroscopy?
- Uv region can be divided into far UV (100-200nm) and near UV (200-400nm)
- During absorption, some molecules of compound collide with the photons of radiant energy
The absorption of radiant energy is based on which laws?
- Beer’s Law = the amount of energy absorbed is directly proportional to the concentration of absorbing solute or number of absorbing molecule present in a medium
- Lambert’s Law = the fraction of light absorbed by a homogenous medium is independent of the intensity of light and light absorption is directly proportional to path length of light
What is the Beer-Lambert Law?
A = log (lo/l) = ε c l
What is Monochromatic light?
Light of a specific single wavelengthength
What is a wavelength?
The distance between consecutive points of a wave
What are the properties of light?
- Violet and blue have shorter wavelengths and higher energy, red wavelengths are longer and have lower energy
- Each type of electromagnetic radiation travels at a particular wavelength
- The longer the wavelength, the less energy is carried; short, tight waves carry the most energy
What are Spectrophotometers?
Measure transmitted light and compute from it the absorption -> can identify which wavelengths of light an organism can absorb
What are the properties of pigments?
- Pigments reflect or transmit the wavelengths they cannot absorb -> appear in the corresponding colour
- Chlorophyll and carotenoids (major photosynthetic pigments)
- Chlorophyll a absorbs wavelengths from either end of the visible spectrum but not green; Carotenoids absorb in the short-wavelength blue region and the yellow, red and orange wavelengths
