Details of experiments between the fragments Q4KWH8~1515-1524 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: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: SKSFV(pThr)TGIR (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
Average holdup BI: 0.71
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 115
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.79
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: 4
Measured pKd: 4.34
Experiment series 2
Protein A protein: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: SKSFV(pThr)TGIR (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
Average holdup BI: 0.56
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.69
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: 4
Measured pKd: 3.98
Experiment series 3
Protein A protein: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: SKSFV(pThr)TGIR (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
Average holdup BI: 0.59
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.58
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: 4.06
Experiment series 4
Protein A protein: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: biotin-ttds-SKSFV(pThr)TGIR (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): 159
Standard deviation of dissociation constant (10-6M): 21
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: 3.8
Experiment series 5
Protein A protein: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: biotin-ttds-SKSFV(pThr)TGIR (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): 136
Standard deviation of dissociation constant (10-6M): 24
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: 3.87