Details of experiments between the fragments P27348~1-245 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: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
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: 0.27
Holdup BI standard deviation: 0.18
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.62
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: 3.45
Experiment series 2
Protein A protein: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
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): 123
Standard deviation of dissociation constant (10-6M): 36
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.08
Number of measurements: 3
Measured pKd: 3.91