Details of experiments between the fragments O43896~1077-1086 and P61981~1-247
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: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 115
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: 1
Measured pKd: 5
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: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 134
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: 1
Measured pKd: 5
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: RYPPY(pThr)TPPR (crude synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): <1
Immobilized partner concentration in holdup experiment (10-6M): 129
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: 1
Measured pKd: 6
Experiment series 4
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: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): 4.8
Standard deviation of dissociation constant (10-6M): 0.5
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.01.18
Number of measurements: 3
Measured pKd: 5.32
Experiment series 5
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: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): 3.5
Standard deviation of dissociation constant (10-6M): 0.4
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.01.10
Number of measurements: 3
Measured pKd: 5.46