Details of experiments between O60292 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: SIPA1L3
Protein A fragment: 1130-1139
Protein A site: 14-3-3_pSer1135
Protein A modification: (SEP)1135
Protein A sequence: SKRPVSFPET
Protein A construct: SKRPV(pSer)FPET (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: 0.64
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): 7.85
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: 4.12
Experiment series 2
Protein A protein: SIPA1L3
Protein A fragment: 1468-1477
Protein A site: 14-3-3_pSer1473
Protein A modification: (SEP)1473
Protein A sequence: RPLPFSDPKK
Protein A construct: RPLPF(pSer)DPKK (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: 0.61
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): 7.58
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: 4.07
Experiment series 3
Protein A protein: SIPA1L3
Protein A fragment: 386-395
Protein A site: 14-3-3_pSer391
Protein A modification: (SEP)391
Protein A sequence: ASRAHSLGGL
Protein A construct: ASRAH(pSer)LGGL (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: 0.64
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): 7.34
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: 4.13
Experiment series 4
Protein A protein: SIPA1L3
Protein A fragment: 167-176
Protein A site: 14-3-3_pSer172
Protein A modification: (SEP)172
Protein A sequence: RHRSSSEITL
Protein A construct: RHRSS(pSer)EITL (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: 0.38
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): 4.66
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: 3.67
Experiment series 5
Protein A protein: SIPA1L3
Protein A fragment: 1377-1386
Protein A site: 14-3-3_pSer1382
Protein A modification: (SEP)1382
Protein A sequence: RPKLYSSGSS
Protein A construct: RPKLY(pSer)SGSS (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: 0.17
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): 4.49
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 6
Protein A protein: SIPA1L3
Protein A fragment: 1539-1548
Protein A site: 14-3-3_pSer1544
Protein A modification: (SEP)1544
Protein A sequence: LQRTLSDESL
Protein A construct: LQRTL(pSer)DESL (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: 0.02
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): 3.26
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: SIPA1L3
Protein A fragment: 92-101
Protein A site: 14-3-3_pSer97
Protein A modification: (SEP)97
Protein A sequence: ALREHSNPSP
Protein A construct: ALREH(pSer)NPSP (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: 0.04
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): 1.68
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: SIPA1L3
Protein A fragment: 141-150
Protein A site: 14-3-3_pSer146
Protein A modification: (SEP)146
Protein A sequence: LSRRRSKDVE
Protein A construct: LSRRR(pSer)KDVE (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: 0.09
Holdup BI standard deviation: 0.38
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): 0.18
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: SIPA1L3
Protein A fragment: 1120-1129
Protein A site: 14-3-3_pSer1125
Protein A modification: (SEP)1125
Protein A sequence: VPFRESQPLH
Protein A construct: VPFRE(pSer)QPLH (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: 0.02
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): 0.13
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 10
Protein A protein: SIPA1L3
Protein A fragment: 1377-1386
Protein A site: 14-3-3_pSer1382
Protein A modification: (SEP)1382
Protein A sequence: RPKLYSSGSS
Protein A construct: biotin-ttds-RPKLY(pSer)SGSS (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): 481
Standard deviation of dissociation constant (10-6M): 22
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: 3.32
Experiment series 11
Protein A protein: SIPA1L3
Protein A fragment: 1377-1386
Protein A site: 14-3-3_pSer1382
Protein A modification: (SEP)1382
Protein A sequence: RPKLYSSGSS
Protein A construct: biotin-ttds-RPKLY(pSer)SGSS (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): 445
Standard deviation of dissociation constant (10-6M): 35
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: 3.35