Details of experiments between P63104 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: 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: 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: 0.86
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): 8.96
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: 4.66
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: 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: 0.98
Holdup BI standard deviation: 0
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): 4.94
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: 5.56
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: 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: 0.05
Holdup BI standard deviation: 0.04
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): 1.25
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: 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: -0.31
Holdup BI standard deviation: 0.72
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): 0.61
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: 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: -0.04
Holdup BI standard deviation: 0.13
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): 0.29
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 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: 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): 2
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.01.19
Number of measurements: 3
Measured pKd: 4.68
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: 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): 14
Standard deviation of dissociation constant (10-6M): 1.2
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.10
Number of measurements: 3
Measured pKd: 4.85