Green Light Lab Report

1. Describe the mechanism by which green fluorescent protein (GFP) is able to emit fluorescent light and why ultraviolet light is required to visualise it. (5 marks)

When GFP is hit with UV light, the chromophore is hit by a photon. This changes the chromophore from ground state (A) to A*, which is a highly excitable state. Due to such a highly excitable state not being able to remain so for very long, the A* state chromophore emits a proton, lowering its state to I*, the energetic I. This I* state chromophore lowers its energy by emitting a photon of green light, lowering its energy to I. However, since I and A have such similar energies, I can convert to A and the photon can simply start the whole process again.

2. GFP is shown below
Recovery is plotted as a function of salt concentration (y axis) and buffer ionic strength (BIS; x axis, running from 0.05 to 0.30 M) at three different pH values for the two salt types. Experimental data points are shown as black dots.

Firstly, the small 300 microliter predictor columns often give higher recoveries than the Tricorn columns, with the minimum value of 66% recovery, even giving good recoveries using smaller salt concentrations and buffer ionic strength, which Tricorn cannot, particularly struggling during pH 5.75, when almost a third of the contour plots gave recoveries 40% or below. In addition, there were more samples taken when using the Predictor columns (9 samples per contour plot), with Tricorn columns only using 5 samples per countour plot.
With both columns, as the pH increased, so did the proportion of high percentage recoveries (>80%). Similarly (in general), as the buffer strength increased, so did recoveries, as well as increased recovery rates with higher concentrations of salt. In addition, both columns found that salt 1 recoveries were higher than salt