Details of experiments between Q9UQC2 and P62258
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: GAB2
Protein A fragment: 128-137
Protein A site: 14-3-3_pSer133
Protein A modification: (SEP)133
Protein A sequence: LRNVSSAGHG
Protein A construct: LRNVS(pSer)AGHG (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Measured dissociation constant (10-6M): <100
Immobilized partner concentration in holdup experiment (10-6M): 143
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: 4
Experiment series 2
Protein A protein: GAB2
Protein A fragment: 386-395
Protein A site: 14-3-3_pThr391
Protein A modification: (TPO)391
Protein A sequence: IPRRNTLPAM
Protein A construct: IPRRN(pThr)LPAM (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.92
Holdup BI standard deviation: 0.02
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): 8.12
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
Measured pKd: 4.91
Experiment series 3
Protein A protein: GAB2
Protein A fragment: 380-389
Protein A site: 14-3-3_pThr385
Protein A modification: (TPO)385
Protein A sequence: RSVAATIPRR
Protein A construct: RSVAA(pThr)IPRR (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.62
Holdup BI standard deviation: 0.05
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): 6.52
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
Measured pKd: 4.06
Experiment series 4
Protein A protein: GAB2
Protein A fragment: 386-395
Protein A site: 14-3-3_pThr391
Protein A modification: (TPO)391
Protein A sequence: IPRRNTLPAM
Protein A construct: IPRRN(pThr)LPAM (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.92
Holdup BI standard deviation: 0.03
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): 6.13
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: 4.97
Experiment series 5
Protein A protein: GAB2
Protein A fragment: 380-389
Protein A site: 14-3-3_pThr385
Protein A modification: (TPO)385
Protein A sequence: RSVAATIPRR
Protein A construct: RSVAA(pThr)IPRR (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.61
Holdup BI standard deviation: 0.03
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): 5.54
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: 4.13
Experiment series 6
Protein A protein: GAB2
Protein A fragment: 205-214
Protein A site: 14-3-3_pSer210
Protein A modification: (SEP)210
Protein A sequence: NARSASFSQG
Protein A construct: NARSA(pSer)FSQG (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.17
Holdup BI standard deviation: 0.06
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): 2.89
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 7
Protein A protein: GAB2
Protein A fragment: 205-214
Protein A site: 14-3-3_pSer210
Protein A modification: (SEP)210
Protein A sequence: NARSASFSQG
Protein A construct: NARSA(pSer)FSQG (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.21
Holdup BI standard deviation: 0.09
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): 2.68
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
Measured pKd: 3.27
Experiment series 8
Protein A protein: GAB2
Protein A fragment: 467-476
Protein A site: 14-3-3_pSer472
Protein A modification: (SEP)472
Protein A sequence: RAGDNSQSVY
Protein A construct: RAGDN(pSer)QSVY (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.12
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): 1.97
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: GAB2
Protein A fragment: 259-268
Protein A site: 14-3-3_pSer264
Protein A modification: (SEP)264
Protein A sequence: TEFRDSTYDL
Protein A construct: TEFRD(pSer)TYDL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.07
Holdup BI standard deviation: 0.09
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): 0.77
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 10
Protein A protein: GAB2
Protein A fragment: 317-326
Protein A site: 14-3-3_pSer322
Protein A modification: (SEP)322
Protein A sequence: PATPLSAYQI
Protein A construct: PATPL(pSer)AYQI (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.06
Holdup BI standard deviation: 0.12
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): 0.52
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 11
Protein A protein: GAB2
Protein A fragment: 259-268
Protein A site: 14-3-3_pSer264
Protein A modification: (SEP)264
Protein A sequence: TEFRDSTYDL
Protein A construct: TEFRD(pSer)TYDL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.19
Holdup BI standard deviation: 0.42
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): 0.41
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 12
Protein A protein: GAB2
Protein A fragment: 207-216
Protein A site: 14-3-3_pSer212
Protein A modification: (SEP)212
Protein A sequence: RSASFSQGTR
Protein A construct: RSASF(pSer)QGTR (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.15
Holdup BI standard deviation: 0.56
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): 0.25
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 13
Protein A protein: GAB2
Protein A fragment: 467-476
Protein A site: 14-3-3_pSer472
Protein A modification: (SEP)472
Protein A sequence: RAGDNSQSVY
Protein A construct: RAGDN(pSer)QSVY (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.01
Holdup BI standard deviation: 0.1
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): 0.09
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: GAB2
Protein A fragment: 317-326
Protein A site: 14-3-3_pSer322
Protein A modification: (SEP)322
Protein A sequence: PATPLSAYQI
Protein A construct: PATPL(pSer)AYQI (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.01
Holdup BI standard deviation: 0.09
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): 0.08
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 15
Protein A protein: GAB2
Protein A fragment: 207-216
Protein A site: 14-3-3_pSer212
Protein A modification: (SEP)212
Protein A sequence: RSASFSQGTR
Protein A construct: biotin-ttds-RSASF(pSer)QGTR (HPLC-purified synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Measured dissociation constant (10-6M): 325
Standard deviation of dissociation constant (10-6M): 73
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.09
Number of measurements: 3
Measured pKd: 3.49