Details of experiments between the fragments P31947~1-248 and Q13136~663-672
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: PPFIA1
Protein B fragment: 663-672
Protein B site: 14-3-3_pSer668
Protein B modification: (SEP)668
Protein B sequence: RVGSGSLDNL
Protein B construct: biotin-ttds-RVGSG(pSer)LDNL (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): below threshold
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.09
Number of measurements: 3
The affinity was below the detection threshold of the assay.
Experiment series 2
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: PPFIA1
Protein B fragment: 663-672
Protein B site: 14-3-3_pSer668
Protein B modification: (SEP)668
Protein B sequence: RVGSGSLDNL
Protein B construct: RVGSG(pSer)LDNL (crude synthetic peptide)
Average holdup BI: 0.13
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.34
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
The affinity was below the detection threshold of the assay.
Experiment series 3
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: PPFIA1
Protein B fragment: 663-672
Protein B site: 14-3-3_pSer668
Protein B modification: (SEP)668
Protein B sequence: RVGSGSLDNL
Protein B construct: RVGSG(pSer)LDNL (crude synthetic peptide)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.8
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
The affinity was below the detection threshold of the assay.
Experiment series 4
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: PPFIA1
Protein B fragment: 663-672
Protein B site: 14-3-3_pSer668
Protein B modification: (SEP)668
Protein B sequence: RVGSGSLDNL
Protein B construct: RVGSG(pSer)LDNL (crude synthetic peptide)
Average holdup BI: 0.17
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.52
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
The affinity was below the detection threshold of the assay.