Supporting Material
An Optimized Firefly Luciferase Bioluminescent Assay
for the Analysis of Free Fatty Acids
Simone M. Marques 1, Luís M. Gonçalves 2, Joaquim C.G. Esteves da Silva*1
Table S1. Experimental designs formulation and set up
Design type and experimental variablesa
Screening: fractional factorialb
Incubation time / minutes
Incubation temperature / oC
ATP concentration ([ATP]) / µM
LUC concentration ([LUC]) / nM
D-LH2 concentration ([ D-LH2]) / µM
Optimization: central compositeb
ATP concentration ([ATP]) / µM
D-LH2 concentration ([ D-LH2]) / µM
a
low
Levels
central
high
0
20 (room)
5
5
5
15
20 (bath)
15
15
15
30
30 (bath)
25
25
25
5
5
15
15
25
25
The response variable was a 1 µM FFA standard mixture in both designs.
b
For the experimental procedure, one replication per experiment was performed, with a total of eleven
testing experiments plus three control experiments in central levels.
Table S2. Center and HOIE significance testing methods results for the fractional factorial screening
design
a
Factor
Center
HOIE
Incubation time / minutes
NS
NS
Incubation temperature / ºC
NS
NS
++
++
LUC concentration ([LUC]) / nM
NS
NS
D-LH2 concentration ([ D-LH2]) / M
++
++
a
Significance of each effect at 95% level: NS, not significant; from + to +++, positive effect; from - to
- - -, negative effect.
Table S3. Analysis of variance (ANOVA) table for the central composite optimization design
SSa
DF
MS
F-ratio p-value B-coefficient
Summary
Model
9.363 x 107 5 1.873 x 107
Error
3.205 x 106 5 6.411 x 105
Adjusted total 9.684 x 107 10 9.684 x 106
Variable
Intercept
[ATP] (A)
a
2.629 x 108
1.113 x 107
29.210
0.0010
1 2.629 x 108 410.030
7
1 1.113 x 10
17.355
0.0000
[D-LH2] (B)
AB
AA
BB
6
6
6.232 x 10
5.233 x 106
4.024 x 107
5.141 x 107
1
1
1
1
6.232 x 10
5.233 x 106
4.024 x 107
5.141 x 107
Model check
Main
Int
Int + squ
Squ
Error
1.736 x 107
5.233 x 106
7.104 x 107
7.104 x 107
3.205 x 106
2
1
2
2
5
8.679 x 106
5.233 x 106
3.552 x 107
3.552 x 107
6.411 x 105
Lack of fit
Lack of fit
Pure error
Total error
3.032 x 106
1.730 x 105
3.205 x 106
3 1.011 x 106
2 8.649 x 104
5 6.411 x 105
SEb
0.0088
9.361 x 103 462.274
117.931 28.308
9.720
8.162
62.770
80.189
0.0263
0.0355
0.0005
0.0003
88.259 28.308
915.000 320.273
-2.136 x 103 269.550
-2.414 x 103 269.550
8.162
55.405
55.405
0.0355
0.0004
0.0004
11.687
0.0798
Maximum point
17.629
16.961
SS, Sum of squares; DF, degrees of freedom; MS, mean squares (ratio between SS and DF); F-ratio, ratio
between ‘between-measures’ MS and ‘within-measures’ (residual) MS; p-value, probability of getting the
F-ratio under the null hypothesis at 95%; B-coefficient, regression coefficient from a multiple linear
regression analysis; SEb, standard error of b.