DLS and SAXS Flashcards
What is Rayleigh Scattering?
When light hits small particles it scatters in all directions.
Particles must be small compared to the wavelength
Some wavelengths scatter more than other
What is observed in the scattering intensity of a laser?
Time dependent fluctuation
Lasers are monochromatic and coherent
Why do you observe a time-dependent fluctuation with a laser?
Because the small molecules are undergoing Brownian motion.
Therefore the distance between scatters is constantly changing
Scattered light then undergoes either constructive or destructive _ due to the surrounding particles
Interference
There is intensity fluctuation within this
What is Mie Scattering?
- Scattering of light or other electromagnetic radiation by particles with similar size or larger than the wavelength of the light
- White light scattered in all directions.
What occurs in opalescent glass and blue eyes?
Rayleigh scattering
Greater for blue light than any other
What is Classical or Static Light Scattering?
Constant measuring of scattering (static)
Measured at various angles
Purposes of Static Light Scattering
- Direct measure of molecular mass
- Determine oligomeric state of protein
- Determine that mass of aggregates
What is dynamic light scattering
Measuring scattering at one angle
Scattering is observed to be changing over time
Also known as photon correlation spectroscopy or quasi-elastic light scattering
Purposes of DLS
- measures rate of diffusion
- Determines “stroke radius” or hydrodynamic radius
- Senses very small amounts of aggregated protein
Do emulsions and molecules in suspension undergo Brownian motion?
Yes
This is the motion induced by the bombardment by solvent molecules that themselves are moving due to their thermal energy
What does the decay time of light scattering fluctuations tell us?
The diffusion coefficient of the particles
Inversely proportional
How to we obtain the decay time?
From the time-dependent correlation function of the scattered light.
How do we determine particle size?
R=(kB T)/6πRη
- D: diffusion constant
- R: hydrodynamic radius
- k: Boltzmann-constant
- T: temperature
- η0: solvent viscosity
Larger particles move _
Slower
Smaller particles move _
Faster
How does DLS work?
- A beam of laser light is focused in the sample
- Particles in the scattering volume scatter light in all directions
- The scattered photons are measured by a photomultiplier tube
- The Intensity appears to fluctuate randomly
- A digital correlator is used to compute the autocorrelation function
What is the autocorrelation function?
It appears as a signal with exponential decay
The bigger the particle the longer the correlation lag time.
So small particles will look like skate ramps (concave) and larger ones will look more convex
Lots of fluctuation = small particle.
What is Pd?
Polydispersity
How much of the proteins formed polymers
More than 20% Pd means that the peak will broaden
What does DLS actually measure?
The hydrodynamic radius
The translational diffusion coefficient will depend not only on the size of the particle “core”, but also on any surface structure
Requirements of DLS
- The particles being measured are ‘spherical’
- The suspension is dilute, such that the scattered light is measured before it is re-scattered by other particles (backscattering).
- The optical properties of the particles and the medium surrounding them is known
- The particles are homogeneous
Applications of DLS
- Protein Crystallography - To check before the crystallization occurs
- Small angle X-ray diffraction
- Nanotechnology
- Medicine
What is Small-Angle X-ray Scattering?
A low-resolution technique that provides high-precision information on size and shape
Useful for flexible systems
Not the same as an X-ray
SAXS is ideal for the characterisation of _
o multidomain proteins (don’t like to crystalize)
o multisubunit complexes
o flexible systems
Scattering can be done with, _, _ or _
X-ray, UV or Infrared radiation.
In X-ray diffraction, crystals have a regular structure with a repeat distance that is about the same as the _
Wavelength
What causes the diffraction pattern in X-ray diffraction?
X-ray interference between wavelengths scattered by atoms within regularly spaced protein in the crystal
What is the theta angle?
The angle of diffraction - measured in degrees
For practical reasons the diffractometer measures an angle twice that of the theta angle – “2-theta”
What can we gain from diffraction patterns?
Can use to calculate electron density map
Position of the reflections tells us something about symmetry
Often see a dark “water ring”
What can we see at 5Å and 1Å resolution?
At low resolution, 5Å or higher one can obtain the shape of the molecule and sometimes identify alpha-helical regions as rods of electron density.
At 1Å resolution one sees atoms as discrete ball of density
Why is small angle scattering observed in molecules in crystals and in solution?
However, small angle scattering arises from the coherent secondary wavelets that are scattered by atoms within a single molecule
Stages of preliminary characterisation
- Purity - SDS page
- Concentration - Absorbance
- Monodispersity - DLS
- Matched solvent - dialyse sample
How to calculated scattering factor?
|s|=4π sinθ/λ = scattering factor
Measure angles for solution then subtract angles from solvent to get scattering factor.
Intensity of scattering becomes _ further from the beam stop.
Less
Stages of data validation
- Initial inspection - Guinier plot
- radiation damage - measure time course
- concentration effects
- standards - measure water and/or secondary protein standard
What is the main sign of radiation damage?
If you measure the signal multiple times and it changes
Reducing conditions can prevent this
What is an indication of an aggregated protein in SAXS?
Steep line in sample-solvent graph, for example graph starts with concave curve.
What does an SAXS log plot tell us?
Shape and size
What does a Guinier plot tell us?
Rg, MM
plots Ln I(s) against s^2
Knowing MM can determine if 24-mer or 48-mer etc
What does a P(r) plot tell us?
Diameter
Usually 1 peak
Maximum y = maximal diameter on x
Can be used to find conformational change
What does a Porod plot tell us?
Volume
(s)*s^4
How can you use SAXS to determine a lo-res 3D model?
Ab initio shape determination
Fill sphere with compact dummy atoms and change their orientation until they match the data for your sample
