13. Data point 15 to 21: add 20 μL of PDZ domain stock (concentration 0.5–2 mM) at each step.
14. Data point 22 to 29: add 60 μL of PDZ domain stock (concentration 0.5–2 mM) at each step.
15. Each PDZ–PBM binding assay is collected in triplicate (using
either biological or technical replicates). Figure 1 shows an
example of the output from a typical titration dataset.
3.4 Data Processing
Data processing requires the calculation of PDZ concentration for
each titration step, [PDZ] n , where n is the individual data point
collected.
1. The PDZ concentration of the first data point is 0: [PDZ] 1 ¼ 0.
PDZ
added
Vstock
( L)
Anisotropy
Trials StdErrAniso (%)
0
0.0283667495155055
3
4.6773
3
0.0274686828061678
3
3.5528
3
0.029768683937325
3
1.898
5
0.0286828152753334
3
5.5035
5
0.0289592691855773
3
1.7806
10
0.0279688320612641
3
4.9895
5
0.0302485839847933
3
3.0321
5
0.0282586258847039
3
1.3758
3
0.0288619028287986
3
1.7269
3
0.0306373436606708
3
3.6168
5
0.0338288561137255
3
3.1001
5
0.0348562383472131
3
1.9562
10
0.0373794594208286
3
2.08
10
0.0405176201406837
3
1.0867
20
0.0473504993123733
3
2.422
20
0.0502904025786218
3
1.5704
20
0.0522171120090482
3
1.3038
20
0.0532442012233099
3
1.3866
20
0.0540631222118147
3
3.3913
20
0.057123332166628
3
4.6715
20
0.0589739543476869
3
2.042
60
0.0641761287443581
3
5.0507
60
0.0657427875852492
3
4.1818
60
0.0689649065572496
3
3.3367
60
0.0693739146349071
3
2.9321
60
0.0709325840527179
3
1.7149
60
0.0716128324888053
3
0.6701
60
0.0738748579212796
3
4.1681
60
0.0735578220264764
3
2.4364
60
0.0757814104680961
3
1.4858
Fig. 1 Sample data output of a PDZ–ligand PBM binding assay. The “PDZ
added V stock ” column shows the volume of stock solution for each data point of
the titration. The “Anisotropy” column shows the average anisotropy value
measured for each titration data point. The “Trials” column shows the number
of measurements used for calculating the average anisotropy. The “StdErrAniso”
column shows the standard deviation of the anisotropy
142
Young Joo Sun and Ernesto J. Fuentes
14. Data point 22 to 29: add 60 μL of PDZ domain stock (concentration 0.5–2 mM) at each step.
15. Each PDZ–PBM binding assay is collected in triplicate (using
either biological or technical replicates). Figure 1 shows an
example of the output from a typical titration dataset.
3.4 Data Processing
Data processing requires the calculation of PDZ concentration for
each titration step, [PDZ] n , where n is the individual data point
collected.
1. The PDZ concentration of the first data point is 0: [PDZ] 1 ¼ 0.
PDZ
added
Vstock
( L)
Anisotropy
Trials StdErrAniso (%)
0
0.0283667495155055
3
4.6773
3
0.0274686828061678
3
3.5528
3
0.029768683937325
3
1.898
5
0.0286828152753334
3
5.5035
5
0.0289592691855773
3
1.7806
10
0.0279688320612641
3
4.9895
5
0.0302485839847933
3
3.0321
5
0.0282586258847039
3
1.3758
3
0.0288619028287986
3
1.7269
3
0.0306373436606708
3
3.6168
5
0.0338288561137255
3
3.1001
5
0.0348562383472131
3
1.9562
10
0.0373794594208286
3
2.08
10
0.0405176201406837
3
1.0867
20
0.0473504993123733
3
2.422
20
0.0502904025786218
3
1.5704
20
0.0522171120090482
3
1.3038
20
0.0532442012233099
3
1.3866
20
0.0540631222118147
3
3.3913
20
0.057123332166628
3
4.6715
20
0.0589739543476869
3
2.042
60
0.0641761287443581
3
5.0507
60
0.0657427875852492
3
4.1818
60
0.0689649065572496
3
3.3367
60
0.0693739146349071
3
2.9321
60
0.0709325840527179
3
1.7149
60
0.0716128324888053
3
0.6701
60
0.0738748579212796
3
4.1681
60
0.0735578220264764
3
2.4364
60
0.0757814104680961
3
1.4858
Fig. 1 Sample data output of a PDZ–ligand PBM binding assay. The “PDZ
added V stock ” column shows the volume of stock solution for each data point of
the titration. The “Anisotropy” column shows the average anisotropy value
measured for each titration data point. The “Trials” column shows the number
of measurements used for calculating the average anisotropy. The “StdErrAniso”
column shows the standard deviation of the anisotropy
142
Young Joo Sun and Ernesto J. Fuentes