Details of experiments between Q04917 and Q13136
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
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: 0.36
Holdup BI standard deviation: 0.03
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): 7.16
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: 3.63
Experiment series 2
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PPFIA1
Protein B fragment: 1154-1163
Protein B site: 14-3-3_pThr1159
Protein B modification: (TPO)1159
Protein B sequence: AGSAETLPAN
Protein B construct: AGSAE(pThr)LPAN (crude synthetic peptide)
Average holdup BI: 0.49
Holdup BI standard deviation: 0.04
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): 7.06
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: 3.87
Experiment series 3
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PPFIA1
Protein B fragment: 525-534
Protein B site: 14-3-3_pSer530
Protein B modification: (SEP)530
Protein B sequence: RPHLGSVPDF
Protein B construct: RPHLG(pSer)VPDF (crude synthetic peptide)
Average holdup BI: 0.56
Holdup BI standard deviation: 0.09
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): 3.64
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: 3.98
Experiment series 4
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PPFIA1
Protein B fragment: 567-576
Protein B site: 14-3-3_pThr572
Protein B modification: (TPO)572
Protein B sequence: PSKVQTLNEQ
Protein B construct: PSKVQ(pThr)LNEQ (crude synthetic peptide)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.03
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): 3.55
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.
Experiment series 5
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PPFIA1
Protein B fragment: 692-701
Protein B site: 14-3-3_pSer697
Protein B modification: (SEP)697
Protein B sequence: SSPPGSGRST
Protein B construct: SSPPG(pSer)GRST (crude synthetic peptide)
Average holdup BI: -0.44
Holdup BI standard deviation: 1.07
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): 0.63
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.
Experiment series 6
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PPFIA1
Protein B fragment: 801-810
Protein B site: 14-3-3_pSer806
Protein B modification: (SEP)806
Protein B sequence: KGIKSSIGRL
Protein B construct: KGIKS(pSer)IGRL (crude synthetic peptide)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.2
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): 0.52
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.
Experiment series 7
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: PPFIA1
Protein B fragment: 758-767
Protein B site: 14-3-3_pSer763
Protein B modification: (SEP)763
Protein B sequence: ALHTVSHEDI
Protein B construct: ALHTV(pSer)HEDI (crude synthetic peptide)
Average holdup BI: -0.15
Holdup BI standard deviation: 0.53
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): 0.3
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.
Experiment series 8
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
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): 324
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: 2022.01.19
Number of measurements: 3
Measured pKd: 3.49