lecture 5 - intro to light and EM radiation Flashcards
How can we determine the concentration of proteins ?What are some techniques?
- Western blotting
- ICC ( Immunocytochemistry )
- ELISA ( enzyme linked immunosorbent assay)
- Spectrophotometer
A western blot
where proteins are separated according to their size, and specific antibodies are used to detect how much of our protein of interest is present.
ICC
where fluorescently tagged antibodies specific to our protein of interest allow visualization with a microscope.
ELISA
enzyme linked immunosorbent assay – eg. commonly used to detect antibodies and other proteins in the blood
Spectrophotometer
this can used for a protein assay to detect how much general protein is present in a sample
what is electromagnetic radiation?
- A type of radiation in which electric and magnetic fields vary simultaneously
- Red - magnetic
- Blue - electric
How is the EM spectrum made up ?
- EM wavelengths vary
What is a high energy wave?
- short wavelength and high frequency
What is a low energy wave?
- long wavelength and low frequency
What type of radiation does NMR use?
Radiowaves
What are ‘waves’?
- waves are regular vibrations that carry energy
What is the equation for Frequency?
V =C/lamda
What is planks law?
There is an inverse relationship between the wavelength and the energy of a wave. The higher the energy, the shorter the wavelength
What is planks equation?
E= hv
E = energy carried by wave h = planks constant v = frequency
What are the wave rules? What happens when two waves in phase with the same amplitude and wavelength combine?
Produces a wave that has twice the amplitude but with same wavelength
Who believed that EM radiation was due to a wave nature?
Maxwell, Hertz and other
Who believed that light has a particulate nature?
Planck and Einstein
Who discovered Wave duality?
Louis de Broglie
How can we use EM absorption to study biomolecules?
- light promotes the molecule to an ‘excited state’ , energy released in heat or light
- Absorption spectroscopy tells you about properties of particular chemical groups
How can we use EM scattering to study biomolecules?
- the light bounces off the molecules
- Scattered light has the same wavelength/energy as the incident radiation, but it changes direction , tells you about shapes
Which techniques use absorption radiation?
- IR spectroscopy
- UV spectroscopy
- CD spectroscopy
- Fluorescence spectroscopy
- NMR spec
Which technique uses scattering radiation?
- Dynamic light scattering
small angle X -ray scattering - x RAY and cry electron microscopy
What is transition?
When biomolecules absorb radiation waves when the wavelength of the radiation matches the distance between energy needed to increase to the next energy level
What is transition doing?
involves excitement of electrons , vibrational energy levels in chemical bonds and also rotational energy levels in single covalent bonds
What is the absorption spectrum?
Detection of how much energy is absorbed
What is the emission spectrum?
Molecule hs to lose the extra energy it has acquired , this is delta E
What happens when EM is directed at a molecule with the same wavelength that matches distance between energy levels?
- The molecule becomes excited
- there is sufficient energy to promote to the next level
What is the rotational energy?
energy levels in a single covalent bond
What is vibrational energy?
energy levels in a chemical bond
What is electronic energy?
energy levels of electrons within atoms
What wavelength can we use to measure molecular vibrations?
infrared
What can molecular vibrations determine?
- functional group
- secondary structure info
- difference spectra
What techniques do electron transitions lead to?
- UV - vis spectroscopy
- Florescence
- circular dichroism
What can UV- vis spectroscopy determine?
- quantification
- 3’ and 4 ‘ structures
What can florescence determine?
- 3˚ and 4˚ structures
- Measuring distances
- Catalytic studies
- Fluorescence microscopy
What can circular dichroism do?
- 2* structure
- protein folding
- protein - protein and protein - nucleic acid interactions
What is the purpose of the entrance slit in spectrophotometer ?
- Where the source of radiation passes in from
What is the purpose of the collimating mirror in a spectrophotometer?
- The radiation is reflected on it, In a parallel beam towards the grating
What does the transmission grating do?
- disperses the radiation into its frequency components , different frequencies reflect from the grating at different angles
what does the focusing mirror do?
- directs the beam onto the image sensor.
What is the General output from a spectroscopy experiment?
Absorbance (or transmittance) vs wavenumber (or wavelength)
