Details of experiments between P63104 and O43524
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 408-417
Protein B site: 14-3-3_pSer413
Protein B modification: (SEP)413
Protein B sequence: MQRSSSFPYT
Protein B construct: MQRSS(pSer)FPYT (crude synthetic peptide)
Average holdup BI: 0.9
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.87
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: 4.82
Experiment series 2
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 316-325
Protein B site: 14-3-3_pSer321
Protein B modification: (SEP)321
Protein B sequence: NASTVSGRLS
Protein B construct: NASTV(pSer)GRLS (crude synthetic peptide)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.28
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: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 550-559
Protein B site: 14-3-3_pSer555
Protein B modification: (SEP)555
Protein B sequence: LSNSVSNMGL
Protein B construct: LSNSV(pSer)NMGL (crude synthetic peptide)
Average holdup BI: 0.14
Holdup BI standard deviation: 0.09
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.75
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: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 227-236
Protein B site: 14-3-3_pSer232
Protein B modification: (SEP)232
Protein B sequence: GTGKSSWWII
Protein B construct: GTGKS(pSer)WWII (crude synthetic peptide)
Average holdup BI: -0.25
Holdup BI standard deviation: 0.37
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.73
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: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 174-183
Protein B site: 14-3-3_pThr179
Protein B modification: (TPO)179
Protein B sequence: PDKRLTLSQI
Protein B construct: PDKRL(pThr)LSQI (crude synthetic peptide)
Average holdup BI: 0.1
Holdup BI standard deviation: 0.17
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.56
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: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 210-219
Protein B site: 14-3-3_pSer215
Protein B modification: (SEP)215
Protein B sequence: IRHNLSLHSR
Protein B construct: IRHNL(pSer)LHSR (crude synthetic peptide)
Average holdup BI: 0.23
Holdup BI standard deviation: 0.4
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.52
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 7
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 248-257
Protein B site: 14-3-3_pSer253
Protein B modification: (SEP)253
Protein B sequence: RRRAVSMDNS
Protein B construct: RRRAV(pSer)MDNS (crude synthetic peptide)
Average holdup BI: -0.23
Holdup BI standard deviation: 1.34
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.12
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 8
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FOXO3
Protein B fragment: 204-213
Protein B site: 14-3-3_pSer209
Protein B modification: (SEP)209
Protein B sequence: AGWKNSIRHN
Protein B construct: AGWKN(pSer)IRHN (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.21
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.03
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.