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Absorption Spectra for Fluorescent Proteins

What is the difference between an absorption spectrum, excitation spectrum, and emissions spectrum?

I deduce the identity of two fluorescent protein (label FP3 and FP6) based on absorption spectrum.

The results of the absorption spectra were plotted in two ways: Y-axis as true absorbance or Y-axis as normalized absorbance. How do the two spectra differ? What information is lost by plotting the Y-axis as normalized absorbance and not true absorbance.

Please help me understand the absorption spectra that I got for my two fluorescent protein.

Please see attached for more detail.


Solution Preview

Absorption spectroscopy is carried out for various reasons, but mostly, a series of absorptions are carried out in order to compare the spectra to eachother (qualitatively), or known concentrations are used to first construct a linear relationship of concentration vs. absorbance, then a sample of unknown concentration can be compared(quantitatively).

In your example, we are speaking of the qualitative sort. You want to compare the general shape of each sample to see if there are any similar features, and also see what differences there are.

Absorbance (your y axis) depends on three factors, the concentration (c), the length of light that passes through your sample (path length, l) and the molar extinction coefficient (usually the Greek symbol e, but we will use simply e). The molar extinction coefficient is a wavelength dependent function, which gives rise to the overall shape of the spectra.

A = ebl (this is what ...

Solution Summary

This solution provides explanations to help understand the absorption spectra for fluorescent proteins.