Details of experiments between Q04917 and O96013
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PAK4
Protein B fragment: 262-271
Protein B site: 14-3-3_pSer267
Protein B modification: (SEP)267
Protein B sequence: LGPHASEPQL
Protein B construct: LGPHA(pSer)EPQL (crude synthetic peptide)
Average holdup BI: 0.53
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.43
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
Measured pKd: 3.93
Experiment series 2
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PAK4
Protein B fragment: 176-185
Protein B site: 14-3-3_pSer181
Protein B modification: (SEP)181
Protein B sequence: DKRPLSGPDV
Protein B construct: DKRPL(pSer)GPDV (crude synthetic peptide)
Average holdup BI: 0.13
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.05
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
The affinity was below the detection threshold of the assay.
Experiment series 3
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PAK4
Protein B fragment: 469-478
Protein B site: 14-3-3_pSer474
Protein B modification: (SEP)474
Protein B sequence: VPRRKSLVGT
Protein B construct: VPRRK(pSer)LVGT (crude synthetic peptide)
Average holdup BI: 0.23
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.78
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
Measured pKd: 3.35
Experiment series 4
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PAK4
Protein B fragment: 36-45
Protein B site: 14-3-3_pSer41
Protein B modification: (SEP)41
Protein B sequence: PRQWQSLIEE
Protein B construct: PRQWQ(pSer)LIEE (crude synthetic peptide)
Average holdup BI: 0.74
Holdup BI standard deviation: 0.11
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.7
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
Measured pKd: 4.34
Experiment series 5
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PAK4
Protein B fragment: 202-211
Protein B site: 14-3-3_pThr207
Protein B modification: (TPO)207
Protein B sequence: GRPFNTYPRA
Protein B construct: GRPFN(pThr)YPRA (crude synthetic peptide)
Average holdup BI: 0.57
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.65
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
Measured pKd: 4.01
Experiment series 6
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PAK4
Protein B fragment: 483-492
Protein B site: 14-3-3_pSer488
Protein B modification: (SEP)488
Protein B sequence: APELISRLPY
Protein B construct: APELI(pSer)RLPY (crude synthetic peptide)
Average holdup BI: 0.05
Holdup BI standard deviation: 0.54
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.07
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
The affinity was below the detection threshold of the assay.