Details of experiments between P62258 and Q9Y4H2
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: IRS2
Protein B fragment: 574-583
Protein B site: 14-3-3_pThr579
Protein B modification: (TPO)579
Protein B sequence: RTYSLTTPAR
Protein B construct: RTYSL(pThr)TPAR (crude synthetic peptide)
Average holdup BI: 0.98
Holdup BI standard deviation: 0.01
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): 8.37
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: 5.52
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: IRS2
Protein B fragment: 574-583
Protein B site: 14-3-3_pThr579
Protein B modification: (TPO)579
Protein B sequence: RTYSLTTPAR
Protein B construct: RTYSL(pThr)TPAR (crude synthetic peptide)
Average holdup BI: 0.96
Holdup BI standard deviation: 0.01
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): 8.11
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: 5.35
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: IRS2
Protein B fragment: 427-436
Protein B site: 14-3-3_pSer432
Protein B modification: (SEP)432
Protein B sequence: HSRSMSMPVA
Protein B construct: HSRSM(pSer)MPVA (crude synthetic peptide)
Average holdup BI: 0.75
Holdup BI standard deviation: 0.03
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): 6.22
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 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: IRS2
Protein B fragment: 718-727
Protein B site: 14-3-3_pSer723
Protein B modification: (SEP)723
Protein B sequence: RTFPASGGGY
Protein B construct: RTFPA(pSer)GGGY (crude synthetic peptide)
Average holdup BI: 0.14
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): 4.4
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 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: IRS2
Protein B fragment: 718-727
Protein B site: 14-3-3_pSer723
Protein B modification: (SEP)723
Protein B sequence: RTFPASGGGY
Protein B construct: RTFPA(pSer)GGGY (crude synthetic peptide)
Average holdup BI: 0.2
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): 3.86
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: 3.25
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: IRS2
Protein B fragment: 379-388
Protein B site: 14-3-3_pSer384
Protein B modification: (SEP)384
Protein B sequence: GARPVSVAGS
Protein B construct: GARPV(pSer)VAGS (crude synthetic peptide)
Average holdup BI: 0.13
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): 3.19
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
Measured pKd: 3.04
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: IRS2
Protein B fragment: 603-612
Protein B site: 14-3-3_pSer608
Protein B modification: (SEP)608
Protein B sequence: ATFSGSAGRL
Protein B construct: ATFSG(pSer)AGRL (crude synthetic peptide)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.01
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): 2.93
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: IRS2
Protein B fragment: 603-612
Protein B site: 14-3-3_pSer608
Protein B modification: (SEP)608
Protein B sequence: ATFSGSAGRL
Protein B construct: ATFSG(pSer)AGRL (crude synthetic peptide)
Average holdup BI: 0.06
Holdup BI standard deviation: 0.03
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): 2.53
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: 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: IRS2
Protein B fragment: 427-436
Protein B site: 14-3-3_pSer432
Protein B modification: (SEP)432
Protein B sequence: HSRSMSMPVA
Protein B construct: HSRSM(pSer)MPVA (crude synthetic peptide)
Average holdup BI: 0.81
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): 2.35
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: 4.57
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: IRS2
Protein B fragment: 341-350
Protein B site: 14-3-3_pSer346
Protein B modification: (SEP)346
Protein B sequence: RSRTDSLAAT
Protein B construct: RSRTD(pSer)LAAT (crude synthetic peptide)
Average holdup BI: 0.08
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): 2.31
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 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: IRS2
Protein B fragment: 379-388
Protein B site: 14-3-3_pSer384
Protein B modification: (SEP)384
Protein B sequence: GARPVSVAGS
Protein B construct: GARPV(pSer)VAGS (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.05
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): 1.72
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: 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: IRS2
Protein B fragment: 1171-1180
Protein B site: 14-3-3_pSer1176
Protein B modification: (SEP)1176
Protein B sequence: RHNSASVENV
Protein B construct: RHNSA(pSer)VENV (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): 1.41
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 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: IRS2
Protein B fragment: 1278-1287
Protein B site: 14-3-3_pSer1283
Protein B modification: (SEP)1283
Protein B sequence: PGDKSSWGRT
Protein B construct: PGDKS(pSer)WGRT (crude synthetic peptide)
Average holdup BI: 0.05
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): 0.83
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 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: IRS2
Protein B fragment: 725-734
Protein B site: 14-3-3_pSer730
Protein B modification: (SEP)730
Protein B sequence: GGYKASSPAE
Protein B construct: GGYKA(pSer)SPAE (crude synthetic peptide)
Average holdup BI: -0.06
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): 0.8
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: 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: IRS2
Protein B fragment: 341-350
Protein B site: 14-3-3_pSer346
Protein B modification: (SEP)346
Protein B sequence: RSRTDSLAAT
Protein B construct: RSRTD(pSer)LAAT (crude synthetic peptide)
Average holdup BI: 0.12
Holdup BI standard deviation: 0.17
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): 0.69
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 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: IRS2
Protein B fragment: 1144-1153
Protein B site: 14-3-3_pSer1149
Protein B modification: (SEP)1149
Protein B sequence: RRRHSSETFS
Protein B construct: RRRHS(pSer)ETFS (crude synthetic peptide)
Average holdup BI: -0.34
Holdup BI standard deviation: 0.57
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): 0.32
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 17
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: IRS2
Protein B fragment: 555-564
Protein B site: 14-3-3_pSer560
Protein B modification: (SEP)560
Protein B sequence: SYRRVSGDAA
Protein B construct: SYRRV(pSer)GDAA (crude synthetic peptide)
Average holdup BI: -0.31
Holdup BI standard deviation: 1.11
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): 0.29
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 18
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: IRS2
Protein B fragment: 555-564
Protein B site: 14-3-3_pSer560
Protein B modification: (SEP)560
Protein B sequence: SYRRVSGDAA
Protein B construct: SYRRV(pSer)GDAA (crude synthetic peptide)
Average holdup BI: -0.12
Holdup BI standard deviation: 0.47
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): 0.23
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 19
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: IRS2
Protein B fragment: 730-739
Protein B site: 14-3-3_pSer735
Protein B modification: (SEP)735
Protein B sequence: SSPAESSPED
Protein B construct: SSPAE(pSer)SPED (crude synthetic peptide)
Average holdup BI: -0.14
Holdup BI standard deviation: 0.54
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): 0.22
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 20
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: IRS2
Protein B fragment: 1159-1168
Protein B site: 14-3-3_pSer1164
Protein B modification: (SEP)1164
Protein B sequence: TPVSPSFAHN
Protein B construct: TPVSP(pSer)FAHN (crude synthetic peptide)
Average holdup BI: 0.01
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): 0.2
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 21
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: IRS2
Protein B fragment: 518-527
Protein B site: 14-3-3_pSer523
Protein B modification: (SEP)523
Protein B sequence: SNTPESIAET
Protein B construct: SNTPE(pSer)IAET (crude synthetic peptide)
Average holdup BI: -0.04
Holdup BI standard deviation: 0.21
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): 0.16
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 22
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: IRS2
Protein B fragment: 1159-1168
Protein B site: 14-3-3_pSer1164
Protein B modification: (SEP)1164
Protein B sequence: TPVSPSFAHN
Protein B construct: TPVSP(pSer)FAHN (crude synthetic peptide)
Average holdup BI: -0.01
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): 0.15
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 23
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: IRS2
Protein B fragment: 725-734
Protein B site: 14-3-3_pSer730
Protein B modification: (SEP)730
Protein B sequence: GGYKASSPAE
Protein B construct: GGYKA(pSer)SPAE (crude synthetic peptide)
Average holdup BI: 0
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): 0.01
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 24
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: IRS2
Protein B fragment: 1278-1287
Protein B site: 14-3-3_pSer1283
Protein B modification: (SEP)1283
Protein B sequence: PGDKSSWGRT
Protein B construct: PGDKS(pSer)WGRT (crude synthetic peptide)
Average holdup BI: 0
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
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 25
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: IRS2
Protein B fragment: 574-583
Protein B site: 14-3-3_pThr579
Protein B modification: (TPO)579
Protein B sequence: RTYSLTTPAR
Protein B construct: biotin-ttds-RTYSL(pThr)TPAR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 9.2
Standard deviation of dissociation constant (10-6M): 1.4
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: 5.04