lecture 5 - intro to light and EM radiation

Question 1

How can we determine the concentration of proteins ?What are some techniques?

Answer 1

• Western blotting
• ICC ( Immunocytochemistry )
• ELISA ( enzyme linked immunosorbent assay)
• Spectrophotometer

Question 2

A western blot

Answer 2

where proteins are separated according to their size, and specific antibodies are used to detect how much of our protein of interest is present.

Question 3

ICC

Answer 3

where fluorescently tagged antibodies specific to our protein of interest allow visualization with a microscope.

Question 4

ELISA

Answer 4

enzyme linked immunosorbent assay – eg. commonly used to detect antibodies and other proteins in the blood

Question 5

Spectrophotometer

Answer 5

this can used for a protein assay to detect how much general protein is present in a sample

Question 6

what is electromagnetic radiation?

Answer 6

• A type of radiation in which electric and magnetic fields vary simultaneously
• Red - magnetic
• Blue - electric

Question 7

How is the EM spectrum made up ?

Answer 7

• EM wavelengths vary

Question 8

What is a high energy wave?

Answer 8

• short wavelength and high frequency

Question 9

What is a low energy wave?

Answer 9

• long wavelength and low frequency

Question 10

What type of radiation does NMR use?

Answer 10

Radiowaves

Question 11

What are ‘waves’?

Answer 11

• waves are regular vibrations that carry energy

Question 12

What is the equation for Frequency?

Answer 12

V =C/lamda

Question 13

What is planks law?

Answer 13

There is an inverse relationship between the wavelength and the energy of a wave. The higher the energy, the shorter the wavelength

Question 14

What is planks equation?

Answer 14

E= hv

E = energy carried by wave 
h = planks constant 
v = frequency

Question 15

What are the wave rules? What happens when two waves in phase with the same amplitude and wavelength combine?

Answer 15

Produces a wave that has twice the amplitude but with same wavelength

Question 16

Who believed that EM radiation was due to a wave nature?

Answer 16

Maxwell, Hertz and other

Question 17

Who believed that light has a particulate nature?

Answer 17

Planck and Einstein

Question 18

Who discovered Wave duality?

Answer 18

Louis de Broglie

Question 19

How can we use EM absorption to study biomolecules?

Answer 19

• light promotes the molecule to an ‘excited state’ , energy released in heat or light
• Absorption spectroscopy tells you about properties of particular chemical groups

Question 20

How can we use EM scattering to study biomolecules?

Answer 20

• 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

Question 21

Which techniques use absorption radiation?

Answer 21

• IR spectroscopy
• UV spectroscopy
• CD spectroscopy
• Fluorescence spectroscopy
• NMR spec

Question 22

Which technique uses scattering radiation?

Answer 22

• Dynamic light scattering
  small angle X -ray scattering
• x RAY and cry electron microscopy

Question 23

What is transition?

Answer 23

When biomolecules absorb radiation waves when the wavelength of the radiation matches the distance between energy needed to increase to the next energy level

Question 24

What is transition doing?

Answer 24

involves excitement of electrons , vibrational energy levels in chemical bonds and also rotational energy levels in single covalent bonds

Question 25

What is the absorption spectrum?

Answer 25

Detection of how much energy is absorbed

Question 26

What is the emission spectrum?

Answer 26

Molecule hs to lose the extra energy it has acquired , this is delta E

Question 27

What happens when EM is directed at a molecule with the same wavelength that matches distance between energy levels?

Answer 27

• The molecule becomes excited

- there is sufficient energy to promote to the next level

Question 28

What is the rotational energy?

Answer 28

energy levels in a single covalent bond

Question 29

What is vibrational energy?

Answer 29

energy levels in a chemical bond

Question 30

What is electronic energy?

Answer 30

energy levels of electrons within atoms

Question 31

What wavelength can we use to measure molecular vibrations?

Answer 31

infrared

Question 32

What can molecular vibrations determine?

Answer 32

• functional group
• secondary structure info
• difference spectra

Question 33

What techniques do electron transitions lead to?

Answer 33

• UV - vis spectroscopy
• Florescence
• circular dichroism

Question 34

What can UV- vis spectroscopy determine?

Answer 34

• quantification

- 3’ and 4 ‘ structures

Question 35

What can florescence determine?

Answer 35

• 3˚ and 4˚ structures
• Measuring distances
• Catalytic studies
• Fluorescence microscopy

Question 36

What can circular dichroism do?

Answer 36

• 2* structure
• protein folding
• protein - protein and protein - nucleic acid interactions

Question 37

What is the purpose of the entrance slit in spectrophotometer ?

Answer 37

• Where the source of radiation passes in from

Question 38

What is the purpose of the collimating mirror in a spectrophotometer?

Answer 38

• The radiation is reflected on it, In a parallel beam towards the grating

Question 39

What does the transmission grating do?

Answer 39

• disperses the radiation into its frequency components , different frequencies reflect from the grating at different angles

Question 40

what does the focusing mirror do?

Answer 40

• directs the beam onto the image sensor.

Question 41

What is the General output from a spectroscopy experiment?

Answer 41

Absorbance (or transmittance) vs wavenumber (or wavelength)