Spectroscopy Flashcards

1
Q

Spectroscopy

A
  • identify biological macromolecules
  • main use: quantification of biological macromolecules
  • how much protein and nucleic acids obtained
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2
Q

Spectrophotometers

A
  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
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3
Q
  1. Take light from polychromatic source and use a monochromator to select a narrow band width of light
A
  • light from light source separated into component wavelength by a diffraction gradient or prism.
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4
Q
  1. Measure relative intensity of light passing through a sample as compared to a control or blank
A
  • 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.
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5
Q

Beer’s law

A
  • 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.
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6
Q

Beer-Lambert Law

A
  • 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)
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7
Q

The molar extinction coefficient

A

is given as a constant and varies for each molecule.

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8
Q

The light absorbed depends on:

A
  • the wavelength
  • the characteristics of the absorbing molecules
  • the concentration of the absorbing molecules.
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9
Q

Comparison of DNA and Protein Absorbance Spectra

A
  • Nucleic acid absorbed at 260 nm

- Protein absorbed at 280 nm

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10
Q

purity ratios

A
  • indication of the purity of a DNA samples

- A260/A280

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11
Q

a pure solution of DNA/RNA

A
  • 1.8
  • 2.0

the addition of protein will typically reduce these values.

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12
Q

Extinction coefficient

A
ds-DNA = 0.02 
ss-DNA = 0.025
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13
Q

Proteins at 280

A
  • 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.
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14
Q

Fluorescent protein spectra

A
  • wavelengths of 400-800
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15
Q

Each instrument has a linear range

A
  • sensitivity of instruments in lab are important

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

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16
Q

if too concentrated

A
  • dilute first then measure

- multiply by dilution factor once you have your reading.

17
Q

Nanodrop

A
  • 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.