Details of experiments between the fragments Q13136~663-672 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: PPFIA1
Protein A fragment: 663-672
Protein A site: 14-3-3_pSer668
Protein A modification: (SEP)668
Protein A sequence: RVGSGSLDNL
Protein A construct: RVGSG(pSer)LDNL (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: 0.41
Holdup BI standard deviation: 0.01
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): 9.32
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: 3.71
Experiment series 2
Protein A protein: PPFIA1
Protein A fragment: 663-672
Protein A site: 14-3-3_pSer668
Protein A modification: (SEP)668
Protein A sequence: RVGSGSLDNL
Protein A construct: RVGSG(pSer)LDNL (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: 0.44
Holdup BI standard deviation: 0.02
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): 8.24
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: 3.79
Experiment series 3
Protein A protein: PPFIA1
Protein A fragment: 663-672
Protein A site: 14-3-3_pSer668
Protein A modification: (SEP)668
Protein A sequence: RVGSGSLDNL
Protein A construct: RVGSG(pSer)LDNL (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: 0.39
Holdup BI standard deviation: 0.04
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): 5.86
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: 3.76
Experiment series 4
Protein A protein: PPFIA1
Protein A fragment: 663-672
Protein A site: 14-3-3_pSer668
Protein A modification: (SEP)668
Protein A sequence: RVGSGSLDNL
Protein A construct: biotin-ttds-RVGSG(pSer)LDNL (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): 255
Standard deviation of dissociation constant (10-6M): 35
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: 3.59
Experiment series 5
Protein A protein: PPFIA1
Protein A fragment: 663-672
Protein A site: 14-3-3_pSer668
Protein A modification: (SEP)668
Protein A sequence: RVGSGSLDNL
Protein A construct: biotin-ttds-RVGSG(pSer)LDNL (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): 125
Standard deviation of dissociation constant (10-6M): 5
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: 3.9