6. (12 points) A fluorescence assay is used to detect green fluorescent protein

Question

6. (12 points) A fluorescence assay is used to detect green fluorescent protein (GFP) in water by exciting samples at 395 nm and measuring the intensity of emission at 509 nm (I). A plot of I versus GFP concentration (in ng/mL) is linear, with a slope of 32.6 I per ng/mL and an intercept of 7.9 I. The standard deviation on the blank is 2.3 intensity units a. An unknown sample gives I 204.2. Calculate the concentration of GFP in ng/mL. b. What is the sensitivity for this assay? e in the fluorometer is replaced by a new source which is 3 times brighter. will If so, by how much? C. this affect the sensitivity of the assay?

Explanation / Answer

6. the fluorescence experiment for GFP protein gave us the following straight line equation,

Intensity (I) = 32.8(concentrtaion of GFP) + 7.9

a. For an unknown sample,

Intensity (I) = 204.2

Feeding this value in the straight line equation,

concentration of GFP protein in the sample = (204.2 - 7.9)/32.6 = 6.0215 ng/ml

b. with standard deviation = 2.3 intensity unit

maxima,

concentration of GFP = [(204.2 + 2.3) - 7.9]/32.6 = 6.0920 ng/ml

miniima,

concentration of GFP = [(204.2 - 2.3) - 7.9]/32.6 = 5.9508 ng/ml

Sensitivity = {[(6.0215 - 5.9508) + (6.0920 - 6.0215)]/2} x 100 = 7.06%

c. If the light source is replaced by a 3 times brighter source, the sensitivity would also increase by the same amount. That is the sensitivity would rise up to 3 times the original value.

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