Details of experiments between O43524 and Q04917
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: FOXO3
Protein A fragment: 408-417
Protein A site: 14-3-3_pSer413
Protein A modification: (SEP)413
Protein A sequence: MQRSSSFPYT
Protein A construct: MQRSS(pSer)FPYT (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.91
Holdup BI standard deviation: 0.03
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): 7.63
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: 4.88
Experiment series 2
Protein A protein: FOXO3
Protein A fragment: 550-559
Protein A site: 14-3-3_pSer555
Protein A modification: (SEP)555
Protein A sequence: LSNSVSNMGL
Protein A construct: LSNSV(pSer)NMGL (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.24
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): 2.19
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: 3.38
Experiment series 3
Protein A protein: FOXO3
Protein A fragment: 174-183
Protein A site: 14-3-3_pThr179
Protein A modification: (TPO)179
Protein A sequence: PDKRLTLSQI
Protein A construct: PDKRL(pThr)LSQI (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.15
Holdup BI standard deviation: 0.12
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): 1.32
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 4
Protein A protein: FOXO3
Protein A fragment: 316-325
Protein A site: 14-3-3_pSer321
Protein A modification: (SEP)321
Protein A sequence: NASTVSGRLS
Protein A construct: NASTV(pSer)GRLS (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.03
Holdup BI standard deviation: 0.03
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): 0.98
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 5
Protein A protein: FOXO3
Protein A fragment: 227-236
Protein A site: 14-3-3_pSer232
Protein A modification: (SEP)232
Protein A sequence: GTGKSSWWII
Protein A construct: GTGKS(pSer)WWII (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.18
Holdup BI standard deviation: 0.3
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): 0.44
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 6
Protein A protein: FOXO3
Protein A fragment: 204-213
Protein A site: 14-3-3_pSer209
Protein A modification: (SEP)209
Protein A sequence: AGWKNSIRHN
Protein A construct: AGWKN(pSer)IRHN (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.08
Holdup BI standard deviation: 0.17
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): 0.39
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.