Details of experiments between O95208 and P62258
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: EPN2
Protein A fragment: 267-276
Protein A site: 14-3-3_pThr272
Protein A modification: (TPO)272
Protein A sequence: QARPQTSGEE
Protein A construct: QARPQ(pThr)SGEE (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Measured dissociation constant (10-6M): <100
Immobilized partner concentration in holdup experiment (10-6M): 116
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 1
Measured pKd: 4
Experiment series 2
Protein A protein: EPN2
Protein A fragment: 168-177
Protein A site: 14-3-3_pSer173
Protein A modification: (SEP)173
Protein A sequence: FGRGSSQPNL
Protein A construct: FGRGS(pSer)QPNL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.52
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.99
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: 3.88
Experiment series 3
Protein A protein: EPN2
Protein A fragment: 168-177
Protein A site: 14-3-3_pSer173
Protein A modification: (SEP)173
Protein A sequence: FGRGSSQPNL
Protein A construct: FGRGS(pSer)QPNL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.46
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.93
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
Measured pKd: 3.87
Experiment series 4
Protein A protein: EPN2
Protein A fragment: 190-199
Protein A site: 14-3-3_pSer195
Protein A modification: (SEP)195
Protein A sequence: GGSPASYHGS
Protein A construct: GGSPA(pSer)YHGS (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.41
Holdup BI standard deviation: 0.1
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.04
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: 3.69
Experiment series 5
Protein A protein: EPN2
Protein A fragment: 190-199
Protein A site: 14-3-3_pSer195
Protein A modification: (SEP)195
Protein A sequence: GGSPASYHGS
Protein A construct: GGSPA(pSer)YHGS (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.19
Holdup BI standard deviation: 0.11
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.51
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 6
Protein A protein: EPN2
Protein A fragment: 403-412
Protein A site: 14-3-3_pSer408
Protein A modification: (SEP)408
Protein A sequence: SVPKNSDPWA
Protein A construct: SVPKN(pSer)DPWA (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.18
Holdup BI standard deviation: 0.32
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.53
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 7
Protein A protein: EPN2
Protein A fragment: 322-331
Protein A site: 14-3-3_pSer327
Protein A modification: (SEP)327
Protein A sequence: KKEHGSLPQQ
Protein A construct: KKEHG(pSer)LPQQ (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.12
Holdup BI standard deviation: 0.43
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.23
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: EPN2
Protein A fragment: 311-320
Protein A site: 14-3-3_pThr316
Protein A modification: (TPO)316
Protein A sequence: ESRRDTVKIP
Protein A construct: ESRRD(pThr)VKIP (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.06
Holdup BI standard deviation: 0.22
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.2
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 9
Protein A protein: EPN2
Protein A fragment: 403-412
Protein A site: 14-3-3_pSer408
Protein A modification: (SEP)408
Protein A sequence: SVPKNSDPWA
Protein A construct: SVPKN(pSer)DPWA (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.02
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.15
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 10
Protein A protein: EPN2
Protein A fragment: 322-331
Protein A site: 14-3-3_pSer327
Protein A modification: (SEP)327
Protein A sequence: KKEHGSLPQQ
Protein A construct: KKEHG(pSer)LPQQ (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.01
Holdup BI standard deviation: 0.33
Immobilized partner concentration in holdup experiment (10-6M): 116
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: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
The affinity was below the detection threshold of the assay.
Experiment series 11
Protein A protein: EPN2
Protein A fragment: 311-320
Protein A site: 14-3-3_pThr316
Protein A modification: (TPO)316
Protein A sequence: ESRRDTVKIP
Protein A construct: ESRRD(pThr)VKIP (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0
Holdup BI standard deviation: 0.11
Immobilized partner concentration in holdup experiment (10-6M): 116
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: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
The affinity was below the detection threshold of the assay.