User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/04: Difference between revisions
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Fluorescence spectroscopy is another way to analyze molecular samples. | Fluorescence spectroscopy is another way to analyze molecular samples. | ||
== | ==Instructions== | ||
Each group will pick a different protein (bovine serum albumin, horseradish peroxidase, pepsin, adenosine deaminase, hemoglobin). You'll need to make 4 samples ranging from 10uM to 0.5uM. Remember, we don't have a lot of most of these proteins, so figure out how to make a stock solution using as little solid protein as possible. The fluorescence cuvette needs only 200uL, so you don't need to make more than that for each concentration that you're measuring. | |||
# Make your solutions | |||
# Take an absorbance measurement of your samples and a blank | |||
# Take a fluorescence measurement of your samples and a blank (excitation: 290nm, emission: 310nm-500nm) | |||
# Analyze your data. (Integrate your corrected spectrum. Integration is MUCH more important in fluorescence spectra than peak height.) | |||
# Normalize your corrected spectra to the absorption intensity at 280nm for the corresponding absorbance spectrum. (That is, divide the fluorescence intensity by the A(280) and plot all of the fluorescence spectra on the same chart. Do they match up? Are they different? What could this mean?) | |||
**Note --- We are only going to have 1 cuvette to use. I expect you to work quickly and spend time analyzing data. | |||
==Notes== | ==Notes== | ||
Revision as of 07:11, 30 August 2013
 Biomaterials Design Lab
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ObjectiveBecome familiar with the fluorescence spectrometer and with protein fluorescence. DescriptionFluorescence spectroscopy is another way to analyze molecular samples. InstructionsEach group will pick a different protein (bovine serum albumin, horseradish peroxidase, pepsin, adenosine deaminase, hemoglobin). You'll need to make 4 samples ranging from 10uM to 0.5uM. Remember, we don't have a lot of most of these proteins, so figure out how to make a stock solution using as little solid protein as possible. The fluorescence cuvette needs only 200uL, so you don't need to make more than that for each concentration that you're measuring.
NotesThis area is for any observations or conclusions that you would like to note.
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