Details of experiments between the fragments P63104~1-245 and O43896~1077-1086
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: 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 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: 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 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: 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