Details of experiments between Q9Y2J2 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: EPB41L3
Protein A fragment: 438-447
Protein A site: 14-3-3_pSer443
Protein A modification: (SEP)443
Protein A sequence: YTMSRSLDGA
Protein A construct: YTMSR(pSer)LDGA (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: 0.76
Holdup BI standard deviation: 0.02
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): 8.6
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: 4.38
Experiment series 2
Protein A protein: EPB41L3
Protein A fragment: 396-405
Protein A site: 14-3-3_pThr401
Protein A modification: (TPO)401
Protein A sequence: PKKFLTLGSK
Protein A construct: PKKFL(pThr)LGSK (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: 0.73
Holdup BI standard deviation: 0.03
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): 7.82
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: 4.31
Experiment series 3
Protein A protein: EPB41L3
Protein A fragment: 415-424
Protein A site: 14-3-3_pSer420
Protein A modification: (SEP)420
Protein A sequence: QTRRASALID
Protein A construct: QTRRA(pSer)ALID (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: 0.1
Holdup BI standard deviation: 0.04
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): 2.71
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 4
Protein A protein: EPB41L3
Protein A fragment: 952-961
Protein A site: 14-3-3_pSer957
Protein A modification: (SEP)957
Protein A sequence: KTETISFGSV
Protein A construct: KTETI(pSer)FGSV (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: 0.11
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): 2.19
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 5
Protein A protein: EPB41L3
Protein A fragment: 455-464
Protein A site: 14-3-3_pSer460
Protein A modification: (SEP)460
Protein A sequence: TTKGISQTNL
Protein A construct: TTKGI(pSer)QTNL (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: 0.04
Holdup BI standard deviation: 0.03
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): 1.97
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
The affinity was below the detection threshold of the assay.
Experiment series 6
Protein A protein: EPB41L3
Protein A fragment: 756-765
Protein A site: 14-3-3_pThr761
Protein A modification: (TPO)761
Protein A sequence: EKRLSTSPVR
Protein A construct: EKRLS(pThr)SPVR (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: -0.27
Holdup BI standard deviation: 1.13
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): 0.21
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: EPB41L3
Protein A fragment: 396-405
Protein A site: 14-3-3_pThr401
Protein A modification: (TPO)401
Protein A sequence: PKKFLTLGSK
Protein A construct: biotin-ttds-PKKFL(pThr)LGSK (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): 30
Standard deviation of dissociation constant (10-6M): 0.6
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: 4.52
Experiment series 8
Protein A protein: EPB41L3
Protein A fragment: 396-405
Protein A site: 14-3-3_pThr401
Protein A modification: (TPO)401
Protein A sequence: PKKFLTLGSK
Protein A construct: biotin-ttds-PKKFL(pThr)LGSK (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): 20
Standard deviation of dissociation constant (10-6M): 0.5
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: 4.71