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Procedure
The procedure for this section of the
experiment is rather simple.
An Agilent 8543
spectrophotometer (pictured above) was used to measure the absorbance of
our samples.
Five-hundred
µL
cuvette holds the sample being measured. In order to blank the
device, a cuvette is filled
with 1X TE buffer. The samples were loaded into the cuvette using a
micro syringe. The absorbance of each sample of green fluorescent
protein was measured. For sake of comparison, the absorbance of
hemoglobin and lysozyme was taken. The absorbance of each sample was
then transferred onto a graph using computer analysis. The final
step is to determine the protein concentration from the absorbance, using
Beer's Law.
Results
The protein concentration was determined using Beer's Law (A=abc),
.where the extinction coefficient was 30,000 (M^-1)(cm^-1) and the path
length = 1mm. At 395nm, the green fluorescent protein
had an absorbance of 2.8028x10^-3, which gives it an concentration (c) of
9.34x10^-7. A second reading was taken to insure accuracy in our
measurement. Also, at 395nm, the absorbance was 2.8596x10^-3, which
gives concentration (c) of 9.532x10^-7.
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