Bio-Rad Fluorescent Assay Kits User Manual
Page 6
Fig. 1. Example plot of standard curve and linear equation.
4.
To calculate the concentration of 4MU released, determine the least
squares regression equation for the line generated by the standard
4MU samples. The equation for a line is y = mx + b, where:
y is the instrument reading (RFU)
x is the sample concentration (nM)
m is the slope of the line
b is the y-intercept
5.
Determine the amount of MUG hydrolyzed in the sample by inserting
the RFU values (y-value) into the linear equation and solving for x.
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4.
Check zero by placing the cuvette containing 1x stop buffer (cuvette 5)
in the instrument. Re-zero the instrument if necessary.
5.
Read the standard curve samples. Record or print relative fluores-
cence units for data interpretation. Fluorescent measurements can be
made continuously. It is not necessary to zero the instrument before
each measurement.
B. Assay Samples
1.
Read samples. Record or print relative fluorescence units for data
interpretation. If sample readings fall outside the standard curve
(RFU value greater than 14,000), reduce the amount of extract in the
reaction and/or shorten the incubation period. If the level of expres-
sion is low, increase the amount of extract in the reaction and/or
lengthen the incubation period.
4.6 Data Analysis
The relative fluorescence units (RFU) are used to compare the activity
level between samples or inserted into the least squares regression equa-
tion to determine the amount of substrate hydrolyzed.
1.
If samples were performed in duplicate or triplicate, use the average
RFU readings.
2.
Subtract the non-transformed RFU values from the samples to cor-
rect for endogenous ß-galactosidase activity.
3.
From the data, prepare a calibration curve by plotting 4-methylum-
belliferone concentration (nM) versus relative fluorescence units
(RFU). An example of the 4MU standard curve is shown in Figure 1.
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16,000
4MU Standard Curve
y = 1.3953x + 64.161
R
2
= 0.9997
4MU (nM)
RFU
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
0
2,000
4,000
6,000
8,000
10,000
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