Details of experiments between P31946 and O95208
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 190-199
Protein B site: 14-3-3_pSer195
Protein B modification: (SEP)195
Protein B sequence: GGSPASYHGS
Protein B construct: GGSPA(pSer)YHGS (crude synthetic peptide)
Average holdup BI: 0.54
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 8.05
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
Measured pKd: 4.07
Experiment series 2
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 168-177
Protein B site: 14-3-3_pSer173
Protein B modification: (SEP)173
Protein B sequence: FGRGSSQPNL
Protein B construct: FGRGS(pSer)QPNL (crude synthetic peptide)
Average holdup BI: 0.66
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.96
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: 4.28
Experiment series 3
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 168-177
Protein B site: 14-3-3_pSer173
Protein B modification: (SEP)173
Protein B sequence: FGRGSSQPNL
Protein B construct: FGRGS(pSer)QPNL (crude synthetic peptide)
Average holdup BI: 0.67
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.88
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: 4.18
Experiment series 4
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 190-199
Protein B site: 14-3-3_pSer195
Protein B modification: (SEP)195
Protein B sequence: GGSPASYHGS
Protein B construct: GGSPA(pSer)YHGS (crude synthetic peptide)
Average holdup BI: 0.47
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.26
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: 3.81
Experiment series 5
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 311-320
Protein B site: 14-3-3_pThr316
Protein B modification: (TPO)316
Protein B sequence: ESRRDTVKIP
Protein B construct: ESRRD(pThr)VKIP (crude synthetic peptide)
Average holdup BI: 0.09
Holdup BI standard deviation: 0.11
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.78
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
The affinity was below the detection threshold of the assay.
Experiment series 6
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 311-320
Protein B site: 14-3-3_pThr316
Protein B modification: (TPO)316
Protein B sequence: ESRRDTVKIP
Protein B construct: ESRRD(pThr)VKIP (crude synthetic peptide)
Average holdup BI: 0.06
Holdup BI standard deviation: 0.09
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.61
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: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 322-331
Protein B site: 14-3-3_pSer327
Protein B modification: (SEP)327
Protein B sequence: KKEHGSLPQQ
Protein B construct: KKEHG(pSer)LPQQ (crude synthetic peptide)
Average holdup BI: 0.16
Holdup BI standard deviation: 0.41
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.31
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: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 403-412
Protein B site: 14-3-3_pSer408
Protein B modification: (SEP)408
Protein B sequence: SVPKNSDPWA
Protein B construct: SVPKN(pSer)DPWA (crude synthetic peptide)
Average holdup BI: 0.03
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.29
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: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 403-412
Protein B site: 14-3-3_pSer408
Protein B modification: (SEP)408
Protein B sequence: SVPKNSDPWA
Protein B construct: SVPKN(pSer)DPWA (crude synthetic peptide)
Average holdup BI: -0.03
Holdup BI standard deviation: 0.12
Immobilized partner concentration in holdup experiment (10-6M): 134
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: 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 10
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: EPN2
Protein B fragment: 322-331
Protein B site: 14-3-3_pSer327
Protein B modification: (SEP)327
Protein B sequence: KKEHGSLPQQ
Protein B construct: KKEHG(pSer)LPQQ (crude synthetic peptide)
Average holdup BI: 0
Holdup BI standard deviation: 0.2
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.01
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.