Details of experiments between Q6IQ23 and P63104
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: PLEKHA7
Protein A fragment: 533-542
Protein A site: 14-3-3_pThr538
Protein A modification: (TPO)538
Protein A sequence: RHGSPTAPI-
Protein A construct: FRHGSP(pThr)API (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 132
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: 1
Measured pKd: 5
Experiment series 2
Protein A protein: PLEKHA7
Protein A fragment: 443-452
Protein A site: 14-3-3_pSer448
Protein A modification: (SEP)448
Protein A sequence: KGDSRSLPLD
Protein A construct: KGDSR(pSer)LPLD (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.81
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 132
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: 4.5
Experiment series 3
Protein A protein: PLEKHA7
Protein A fragment: 560-569
Protein A site: 14-3-3_pSer565
Protein A modification: (SEP)565
Protein A sequence: VPRSISVPPS
Protein A construct: VPRSI(pSer)VPPS (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.87
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 8.17
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: 4.69
Experiment series 4
Protein A protein: PLEKHA7
Protein A fragment: 1008-1017
Protein A site: 14-3-3_pThr1013
Protein A modification: (TPO)1013
Protein A sequence: ESRYQTLPGR
Protein A construct: ESRYQ(pThr)LPGR (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.66
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.66
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: 4.17
Experiment series 5
Protein A protein: PLEKHA7
Protein A fragment: 1032-1041
Protein A site: 14-3-3_pThr1037
Protein A modification: (TPO)1037
Protein A sequence: IAPYVTLRRG
Protein A construct: IAPYV(pThr)LRRG (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.28
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.47
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: 3.46
Experiment series 6
Protein A protein: PLEKHA7
Protein A fragment: 981-990
Protein A site: 14-3-3_pSer986
Protein A modification: (SEP)986
Protein A sequence: LRSYVSEPEL
Protein A construct: LRSYV(pSer)EPEL (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.59
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.42
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: 4.04
Experiment series 7
Protein A protein: PLEKHA7
Protein A fragment: 467-476
Protein A site: 14-3-3_pThr472
Protein A modification: (TPO)472
Protein A sequence: PENYQTLPKS
Protein A construct: PENYQ(pThr)LPKS (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.2
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.87
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: PLEKHA7
Protein A fragment: 1105-1114
Protein A site: 14-3-3_pSer1110
Protein A modification: (SEP)1110
Protein A sequence: SSRYLSRPLP
Protein A construct: SSRYL(pSer)RPLP (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.21
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.77
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 9
Protein A protein: PLEKHA7
Protein A fragment: 897-906
Protein A site: 14-3-3_pThr902
Protein A modification: (TPO)902
Protein A sequence: QLRKVTSPLQ
Protein A construct: QLRKV(pThr)SPLQ (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.14
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.59
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: PLEKHA7
Protein A fragment: 626-635
Protein A site: 14-3-3_pSer631
Protein A modification: (SEP)631
Protein A sequence: HVDRRSMPSM
Protein A construct: HVDRR(pSer)MPSM (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.24
Holdup BI standard deviation: 0.11
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.24
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: 3.37
Experiment series 11
Protein A protein: PLEKHA7
Protein A fragment: 599-608
Protein A site: 14-3-3_pSer604
Protein A modification: (SEP)604
Protein A sequence: PDQRRSVDIS
Protein A construct: PDQRR(pSer)VDIS (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.05
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.13
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: PLEKHA7
Protein A fragment: 603-612
Protein A site: 14-3-3_pSer608
Protein A modification: (SEP)608
Protein A sequence: RSVDISLGDS
Protein A construct: RSVDI(pSer)LGDS (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: -0.06
Holdup BI standard deviation: 0.09
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.75
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: PLEKHA7
Protein A fragment: 1058-1067
Protein A site: 14-3-3_pSer1063
Protein A modification: (SEP)1063
Protein A sequence: LERLYSGDHQ
Protein A construct: LERLY(pSer)GDHQ (crude synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Average holdup BI: 0.01
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.12
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: PLEKHA7
Protein A fragment: 443-452
Protein A site: 14-3-3_pSer448
Protein A modification: (SEP)448
Protein A sequence: KGDSRSLPLD
Protein A construct: biotin-ttds-KGDSR(pSer)LPLD (HPLC-purified synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Measured dissociation constant (10-6M): 100
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: 2022.02.10
Number of measurements: 3
Measured pKd: 4
Experiment series 15
Protein A protein: PLEKHA7
Protein A fragment: 443-452
Protein A site: 14-3-3_pSer448
Protein A modification: (SEP)448
Protein A sequence: KGDSRSLPLD
Protein A construct: biotin-ttds-KGDSR(pSer)LPLD (HPLC-purified synthetic peptide)
Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta
Measured dissociation constant (10-6M): 98
Standard deviation of dissociation constant (10-6M): 4
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: 4.01