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MEASUREMENT OF SERUM CHOLESTEROL USING A FLUORESCENCE ASSAY
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Worksheet EXPERIMENT 3.3: MEASUREMENTOF SERUM CHOLESTEROL USING A
FLUORESCENCE ASSAY
25 November 2020
08:37
Aim of the · To determine the cholesterol content of a serum sample
experiment
Your learning · To use fluorimetry to determine the concentration of a
objectives medically important constituent of blood
· To gain experience in using a coupled assay
· To gain experience in the use of enzymes for assaying the
concentration of metabolites
SESSION ORGANISATION
All timings are approximate.
10:00-10:30: Make sure you arrive in good time. Download a copy of the session worksheet
from Minerva before or as soon as you arrive. You will enter the breakout room with your
group. Check your answers to Q1-4 with one another and with your demonstrator
10:30–11:00: Brief introduction by the academic lead together with any course-related
announcements.
11:00-12:45 (including 15 mins coffee/tea breaks 11:30-11:45): You will enter the
breakout room with your group. Complete worksheet in your groups with help from your
demonstrator. Remember that while you are working as a group, each of you must complete
their own worksheet, in your own words.
12:45-13:00: Conclusion and questions by the academic lead.
Q1-4
Discuss the following questions within your group – be prepared to explain your reasoning
and support one another Check your answers with your demonstrator before
proceeding to the next section.
Q1: Give few examples of how fluorescence can be used in biological studies?
, Changes in fluorescence intensity can be used to probe structural changes or binding of two
molecules. The wavelength of tryptophan(has a natural fluorescence) fluorescence can be used to
determine whether a tryptophan is in an aqueous environment (longer wavelength) or buried deep
within the protein (shorter wavelength). Fluorescence polarization anisotropy allows mobility of
fluorophores to be studies.
APPLICATION:
1. FRET ,
2. fluorescent antibody labelling
3. Find where fluorescent is positioned.
4. Track substrate changing colour ( fluorogenic substrate)
chemical sensors (fluorescence spectroscopy),
fluorescent labelling,
dyes,
From <https://warwick.ac.uk/services/ris/impactinnovation/impact/analyticalguide/fluorescence>
Q2: How is fluorescence generated?
Fluorescence occurs when a fluorescent capable material (a fluorophore) is excited into a higher
electronic state by absorbing an incident photon and cannot return to the ground state except by
emitting a photon. (then release a photon of a lower wavelength)The emission usually occurs from
the ground vibrational level of the excited electronic state and goes to an excited vibrational state of
the ground electronic state. Thus fluorescence signals occur at longer wavelengths than absorbance.
The energies and relative intensities of the fluorescence signals give information about structure and
environments of the fluorophores.
Q3: What is the difference between excitation and emission wavelength?
Excitation wavelength is shorter than emission wavelength because it has a higher energy
Q4: What kind chemical structures do molecules with fluorescence contain?
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound
that can re-emit light upon light excitation. Fluorophores typically contain several combined
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