Details of experiments between the fragments P63104~1-245 and Q9Y4H2~574-583
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: IRS2
Protein B fragment: 574-583
Protein B site: 14-3-3_pThr579
Protein B modification: (TPO)579
Protein B sequence: RTYSLTTPAR
Protein B construct: RTYSL(pThr)TPAR (crude synthetic peptide)
Measured dissociation constant (10-6M): <1
Immobilized partner concentration in holdup experiment (10-6M): 132
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 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: IRS2
Protein B fragment: 574-583
Protein B site: 14-3-3_pThr579
Protein B modification: (TPO)579
Protein B sequence: RTYSLTTPAR
Protein B construct: biotin-ttds-RTYSL(pThr)TPAR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 6.3
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: 2022.01.19
Number of measurements: 3
Measured pKd: 5.2
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: IRS2
Protein B fragment: 574-583
Protein B site: 14-3-3_pThr579
Protein B modification: (TPO)579
Protein B sequence: RTYSLTTPAR
Protein B construct: biotin-ttds-RTYSL(pThr)TPAR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 4.6
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: 2022.02.10
Number of measurements: 3
Measured pKd: 5.33