Details of experiments between the fragments P63104~1-245 and Q9UQC2~207-216
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: GAB2
Protein B fragment: 207-216
Protein B site: 14-3-3_pSer212
Protein B modification: (SEP)212
Protein B sequence: RSASFSQGTR
Protein B construct: RSASF(pSer)QGTR (crude synthetic peptide)
Average holdup BI: 0.39
Holdup BI standard deviation: 0.08
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): 4.34
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: 3.68
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: GAB2
Protein B fragment: 207-216
Protein B site: 14-3-3_pSer212
Protein B modification: (SEP)212
Protein B sequence: RSASFSQGTR
Protein B construct: biotin-ttds-RSASF(pSer)QGTR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 195
Standard deviation of dissociation constant (10-6M): 7
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: 3.71
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: GAB2
Protein B fragment: 207-216
Protein B site: 14-3-3_pSer212
Protein B modification: (SEP)212
Protein B sequence: RSASFSQGTR
Protein B construct: biotin-ttds-RSASF(pSer)QGTR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 166
Standard deviation of dissociation constant (10-6M): 53
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: 3.78