Details of experiments between the fragments O43896~1077-1086 and P62258~1-255
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: KIF1C
Protein A fragment: 1077-1086
Protein A site: 14-3-3_pThr1082
Protein A modification: (TPO)1082
Protein A sequence: RYPPYTTPPR
Protein A construct: RYPPY(pThr)TPPR (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: 0.98
Holdup BI standard deviation: 0.02
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): 6.52
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: 5.55
Experiment series 2
Protein A protein: KIF1C
Protein A fragment: 1077-1086
Protein A site: 14-3-3_pThr1082
Protein A modification: (TPO)1082
Protein A sequence: RYPPYTTPPR
Protein A construct: RYPPY(pThr)TPPR (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: 0.89
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): 4.03
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: 4.76
Experiment series 3
Protein A protein: KIF1C
Protein A fragment: 1077-1086
Protein A site: 14-3-3_pThr1082
Protein A modification: (TPO)1082
Protein A sequence: RYPPYTTPPR
Protein A construct: biotin-ttds-RYPPY(pThr)TPPR (HPLC-purified 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): 17
Standard deviation of dissociation constant (10-6M): 3
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: 4.76