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Spectrofluorometer

1. The document outlines procedures for measuring the fluorescence excitation and emission spectrum of fluorescein using a fluorescence spectrometer. This includes taking excitation and emission scans of fluorescein at different concentrations and excitation wavelengths. 2. Steps are provided to measure the fluorescence anisotropy of fluorescein by using a polarizer in the excitation and emission paths, calibrating the G-factor, and measuring the vertical and horizontal emission fluorescence of fluorescein alone and after adding 4420 antibody. 3. Comparisons are made between fluorescence spectra and anisotropy measurements of fluorescein at different concentrations and with and without the 4420 antibody.

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175 views2 pages

Spectrofluorometer

1. The document outlines procedures for measuring the fluorescence excitation and emission spectrum of fluorescein using a fluorescence spectrometer. This includes taking excitation and emission scans of fluorescein at different concentrations and excitation wavelengths. 2. Steps are provided to measure the fluorescence anisotropy of fluorescein by using a polarizer in the excitation and emission paths, calibrating the G-factor, and measuring the vertical and horizontal emission fluorescence of fluorescein alone and after adding 4420 antibody. 3. Comparisons are made between fluorescence spectra and anisotropy measurements of fluorescein at different concentrations and with and without the 4420 antibody.

Uploaded by

ctza88
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Measure the fluorescence excitation and emission spectrum

1. Turn on the instrument


a. Turn on the lamp power
b. Turn on the computer, monochromater, detector.
c. Open the software Felix
2. Excitation measurement procedure
a. In the menu ‘Acquisition’, select ‘New Acquisition/excitation scan’
b. Choose the appropriate excitation wavelength range and the emission
wavelength. (The emission and excitation wavelength cannot overlap)
c. Click the button ‘More’, select ‘Real time spectrum correction’ and select
both ‘Excitation correction’ and ‘emission correction’
d. Click ‘Acquire’, then ‘Start’
3. Emission measurement procedure
a. In the menu ‘Acquisition’, select ‘New Acquisition/Emission scan’
b. Choose the appropriate emission wavelength range and the excitation
wavelength. (The emission and excitation wavelength cannot overlap)
c. Click the button ‘More’, select ‘Real time spectrum correction’ and select
both ‘Excitation correction’ and ‘emission correction’
d. Click ‘Acquire’, then ‘Start’
4. Measure Fluorescein excitation and emission spectrum
a. Measure the buffer excitation spectrum (excitation: 400nm-510nm,
emission: 530nm) and emission spectrum (excitation: 460nm, emission:
480nm-600nm)
b. Dilute the fluorescein stock solution by a factor 100 x 50 (16nM)
c. Measure the excitation and emission spectra using the same settings as in
a.
d. Subtract the measured fluorescein spectrum from the background
spectrum, normalize; you now have the corrected spectrum.
e. Now take the emission spectrum at a different excitation wavelength
(440nm). Compare the emission spectra at different excitation
wavelengths.
f. Dilute the fluorescein stock solution by a factor of 100x100 (8nm),
measure the emission spectrum. Compare the fluorescence spectra and
show that the concentration and fluorescence signal are proportional to
each other.
5. Measure Fluorescence anisotropy
a. Put the polarizer in the excitation and emission path
b. In the ‘Acquisition’, select ‘New Acquisition/Emission Scan with
Polarizer’. Choose appropriate excitation and emission wavelengths for
fluorescein.
c. Calibrate the G-factor: Use horizontal excitation light, measure vertical
and horizontal emission fluorescence. Take the ratio between vertical and
horizontal fluorescence.
d. Measure the anisotropy of the fluorescein sample. Use vertical excitation
light, measure the vertical and horizontal emission fluorescence. Calculate
the anisotropy.
e. Add 4420 antibody and measure the anisotropy.

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