Details of experiments between P62258 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: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: 0.15
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.88
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: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: 0.18
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.27
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: 3.19
Experiment series 4
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: 0.27
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.27
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: 3.41
Experiment series 5
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: 0.26
Holdup BI standard deviation: 0.12
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.03
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: 3.49
Experiment series 6
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: 0.27
Holdup BI standard deviation: 0.13
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.97
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: 3.41
Experiment series 7
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: -0.09
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.4
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 8
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: 0.23
Holdup BI standard deviation: 0.17
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.08
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 9
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: 0.03
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.8
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 10
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: -0.23
Holdup BI standard deviation: 0.53
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.56
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 11
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: -0.12
Holdup BI standard deviation: 0.23
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.49
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 12
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Protein B protein: PPFIA1
Protein B fragment: 735-744
Protein B site: 14-3-3_pThr740
Protein B modification: (TPO)740
Protein B sequence: TIKCETSPPS
Protein B construct: TIKCE(pThr)SPPS (crude synthetic peptide)
Average holdup BI: -0.3
Holdup BI standard deviation: 1.29
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.24
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
The affinity was below the detection threshold of the assay.
Experiment series 13
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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: -0.22
Holdup BI standard deviation: 1.09
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.19
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 14
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Protein B protein: PPFIA1
Protein B fragment: 1134-1143
Protein B site: 14-3-3_pSer1139
Protein B modification: (SEP)1139
Protein B sequence: FRRAPSWRKK
Protein B construct: FRRAP(pSer)WRKK (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.69
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.08
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 15
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Protein B protein: PPFIA1
Protein B fragment: 695-704
Protein B site: 14-3-3_pSer700
Protein B modification: (SEP)700
Protein B sequence: PGSGRSTPRR
Protein B construct: PGSGR(pSer)TPRR (crude synthetic peptide)
Average holdup BI: -0.05
Holdup BI standard deviation: 0.94
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.04
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 16
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
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
Holdup BI standard deviation: 0.1
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.03
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.