Details of experiments between the fragments Q9P2M7~126-135 and P63104~1-245
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: CGN
Protein A fragment: 126-135
Protein A site: 14-3-3_pSer131
Protein A modification: (SEP)131
Protein A sequence: LLRSHSQASL
Protein A construct: LLRSH(pSer)QASL (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.24
Holdup BI standard deviation: 0.06
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): 3.74
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: 3.37
Experiment series 2
Protein A protein: CGN
Protein A fragment: 126-135
Protein A site: 14-3-3_pSer131
Protein A modification: (SEP)131
Protein A sequence: LLRSHSQASL
Protein A construct: biotin-ttds-LLRSH(pSer)QASL (HPLC-purified synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Measured dissociation constant (10-6M): 105
Standard deviation of dissociation constant (10-6M): 2
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: 3.98
Experiment series 3
Protein A protein: CGN
Protein A fragment: 126-135
Protein A site: 14-3-3_pSer131
Protein A modification: (SEP)131
Protein A sequence: LLRSHSQASL
Protein A construct: biotin-ttds-LLRSH(pSer)QASL (HPLC-purified synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Measured dissociation constant (10-6M): 96
Standard deviation of dissociation constant (10-6M): 30
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: 4.02