235
K x PO 4 (pH 5.1): 1 M KH 2 PO 4 , adjust pH value to 5.1
using 2 M KOH (use approximately 15 ml for 1 l). Adjust pH
value to 5.0. Add coelenterazin afterwards.
For storage, make 50 ml aliquots and store at −20 °C in
the dark. Aliquots can be stored up to 6 months.
15. The package ‘drc’ is used in R to calculate EC50 and IC50
values and to generate fitted dose response curves. The script
uses the drm function and is amended to include error bars
based on the standard error of the mean (sem). The assay data
is saved as txt file using a tab delimited format. Luciferase activity (activity) is calculated in %, concentration (conc) in μM,
and sem in %.
A sample assay data set is shown below:
activity
conc
sem
7.024253731
0.00003
0.969091058
8.789256841
0.0001
1.875653638
10.69690609
0.0003
1.271332669
7.650808458
0.001
0.991970081
12.68326337
0.003
1.117476168
9.120219216
0.01
1.537416736
9.865127488
0.03
1.367412567
13.02510883
0.1
2.670494192
26.97566853
0.3
3.160663768
31.31743626
1
2.324396584
61.8740283
3
3.084176905
70.77386505
10
9.107073314
100
30
5.429347986
95.9036847
100
13.04036002
The drc script including the addition of error bars reads:
# Loading package
library("drc")
# Loading data
drc <- read.delim ("D:/…/DRC_1.txt”, header=T,
dec=".")
# Returning data
drc
# Fitting the model
DR.1 <- drm(activity~conc, data = drc, fct =
LL.4())
summary(DR.1)
Characterising Dynamic PPIs Using Split TEV