Details of experiments between P31947 and O43896
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 1077-1086
Protein B site: 14-3-3_pThr1082
Protein B modification: (TPO)1082
Protein B sequence: RYPPYTTPPR
Protein B construct: RYPPY(pThr)TPPR (crude synthetic peptide)
Average holdup BI: 0.93
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 8.23
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: 5.1
Experiment series 2
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 1087-1096
Protein B site: 14-3-3_pSer1092
Protein B modification: (SEP)1092
Protein B sequence: MRRQRSAPDL
Protein B construct: MRRQR(pSer)APDL (crude synthetic peptide)
Average holdup BI: 0.61
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.37
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: 4.05
Experiment series 3
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 1077-1086
Protein B site: 14-3-3_pThr1082
Protein B modification: (TPO)1082
Protein B sequence: RYPPYTTPPR
Protein B construct: RYPPY(pThr)TPPR (crude synthetic peptide)
Average holdup BI: 0.93
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.36
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: 4.94
Experiment series 4
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 1087-1096
Protein B site: 14-3-3_pSer1092
Protein B modification: (SEP)1092
Protein B sequence: MRRQRSAPDL
Protein B construct: MRRQR(pSer)APDL (crude synthetic peptide)
Average holdup BI: 0.54
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5
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: 4.02
Experiment series 5
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 1087-1096
Protein B site: 14-3-3_pSer1092
Protein B modification: (SEP)1092
Protein B sequence: MRRQRSAPDL
Protein B construct: MRRQR(pSer)APDL (crude synthetic peptide)
Average holdup BI: 0.6
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.57
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: 4.12
Experiment series 6
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 1077-1086
Protein B site: 14-3-3_pThr1082
Protein B modification: (TPO)1082
Protein B sequence: RYPPYTTPPR
Protein B construct: RYPPY(pThr)TPPR (crude synthetic peptide)
Average holdup BI: 0.9
Holdup BI standard deviation: 0.13
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.33
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: 4.89
Experiment series 7
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 484-493
Protein B site: 14-3-3_pThr489
Protein B modification: (TPO)489
Protein B sequence: REDGGTVGVF
Protein B construct: REDGG(pThr)VGVF (crude synthetic peptide)
Average holdup BI: 0.06
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.6
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 8
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 484-493
Protein B site: 14-3-3_pThr489
Protein B modification: (TPO)489
Protein B sequence: REDGGTVGVF
Protein B construct: REDGG(pThr)VGVF (crude synthetic peptide)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.04
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 9
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 669-678
Protein B site: 14-3-3_pSer674
Protein B modification: (SEP)674
Protein B sequence: RLYADSDSGD
Protein B construct: RLYAD(pSer)DSGD (crude synthetic peptide)
Average holdup BI: -0.33
Holdup BI standard deviation: 0.71
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.67
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: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 272-281
Protein B site: 14-3-3_pThr277
Protein B modification: (TPO)277
Protein B sequence: NKSLTTLGKV
Protein B construct: NKSLT(pThr)LGKV (crude synthetic peptide)
Average holdup BI: -0.02
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.35
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: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 669-678
Protein B site: 14-3-3_pSer674
Protein B modification: (SEP)674
Protein B sequence: RLYADSDSGD
Protein B construct: RLYAD(pSer)DSGD (crude synthetic peptide)
Average holdup BI: -0.19
Holdup BI standard deviation: 0.61
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.3
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 12
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 669-678
Protein B site: 14-3-3_pSer674
Protein B modification: (SEP)674
Protein B sequence: RLYADSDSGD
Protein B construct: RLYAD(pSer)DSGD (crude synthetic peptide)
Average holdup BI: 0.05
Holdup BI standard deviation: 0.16
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.25
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 13
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 484-493
Protein B site: 14-3-3_pThr489
Protein B modification: (TPO)489
Protein B sequence: REDGGTVGVF
Protein B construct: REDGG(pThr)VGVF (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.24
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 14
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 272-281
Protein B site: 14-3-3_pThr277
Protein B modification: (TPO)277
Protein B sequence: NKSLTTLGKV
Protein B construct: NKSLT(pThr)LGKV (crude synthetic peptide)
Average holdup BI: -0.02
Holdup BI standard deviation: 0.12
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.14
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 15
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 272-281
Protein B site: 14-3-3_pThr277
Protein B modification: (TPO)277
Protein B sequence: NKSLTTLGKV
Protein B construct: NKSLT(pThr)LGKV (crude synthetic peptide)
Average holdup BI: -0.02
Holdup BI standard deviation: 0.12
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.13
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 16
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: KIF1C
Protein B fragment: 1077-1086
Protein B site: 14-3-3_pThr1082
Protein B modification: (TPO)1082
Protein B sequence: RYPPYTTPPR
Protein B construct: biotin-ttds-RYPPY(pThr)TPPR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 21
Standard deviation of dissociation constant (10-6M): 4
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.09
Number of measurements: 3
Measured pKd: 4.67