Spectroscopy

Question 1

Spectroscopy

Answer 1

• identify biological macromolecules
• main use: quantification of biological macromolecules
• how much protein and nucleic acids obtained

Question 2

Spectrophotometers

Answer 2

1. Take light from polychromatic source and use a monochromator to select a narrow band width of light
2. Measure relative intensity of light passing through a sample as compared to a control or blank

Question 3

1. Take light from polychromatic source and use a monochromator to select a narrow band width of light

Answer 3

• light from light source separated into component wavelength by a diffraction gradient or prism.

Question 4

2. Measure relative intensity of light passing through a sample as compared to a control or blank

Answer 4

• monochromatic light passed through a cuvette containing protein or nucleic acid being measured and then transmitted light is measured by a detector displayed on a spectrophotometer
• measure relative light intensity of beam before and after it passes through the test sample.
• light that comes through sample has to be compared to a control or blank that consists of the solution (water or buffer) the sample is in.
  
  • ensures quantification of only the biological molecule of interest.

Question 5

Beer’s law

Answer 5

• the amount of light transmitted is inversely proportional to the concentration of molecules in a sample
• more molecules in a sample to absorb the light, the less that will move through the sample and be detected.

Question 6

Beer-Lambert Law

Answer 6

• there is a linear relationship between the absorbance and the concentration

A=Elc

A = measure of absorbance (no units)
E = molar extinction coefficient or absorption coefficient (L/mol*cm)
l = is the path length (cm)
c = the concentration (mol/L) or (ug/mL)

Question 7

The molar extinction coefficient

Answer 7

is given as a constant and varies for each molecule.

Question 8

The light absorbed depends on:

Answer 8

• the wavelength
• the characteristics of the absorbing molecules
• the concentration of the absorbing molecules.

Question 9

Comparison of DNA and Protein Absorbance Spectra

Answer 9

• Nucleic acid absorbed at 260 nm

- Protein absorbed at 280 nm

Question 10

purity ratios

Answer 10

• indication of the purity of a DNA samples

- A260/A280

Question 11

a pure solution of DNA/RNA

Answer 11

• 1.8
• 2.0

the addition of protein will typically reduce these values.

Question 12

Extinction coefficient

Answer 12

ds-DNA = 0.02 
ss-DNA = 0.025

Question 13

Proteins at 280

Answer 13

• The Protein A280 method is applicable to puried proteins that contain Trp, Tyr, or Phe residues
• Trp>Tyr>Phe
• helps to know AA composition of a protein when quantifying. Will have impact on extinction coefficient of your particular protein.

Question 14

Fluorescent protein spectra

Answer 14

• wavelengths of 400-800

Question 15

Each instrument has a linear range

Answer 15

• sensitivity of instruments in lab are important

- samples too concentrated or dilute will give an incorrect value on spec.

Question 16

if too concentrated

Answer 16

• dilute first then measure

- multiply by dilution factor once you have your reading.

Question 17

Nanodrop

Answer 17

• use a small amount of sample (~1 mL) to quantify
• most traditional specs require 1 mL or 100 mL
• normally connected to a computer that does calculation for you.