Details of experiments between Q4KWH8 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: PLCH1
Protein A fragment: 1625-1634
Protein A site: 14-3-3_pSer1630
Protein A modification: (SEP)1630
Protein A sequence: AVNRHSTGSY
Protein A construct: AVNRH(pSer)TGSY (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): 116
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: 1
Measured pKd: 4
Experiment series 2
Protein A protein: PLCH1
Protein A fragment: 965-974
Protein A site: 14-3-3_pSer970
Protein A modification: (SEP)970
Protein A sequence: QARPVSMPVD
Protein A construct: QARPV(pSer)MPVD (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: 0.74
Holdup BI standard deviation: 0.13
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): 4.93
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: PLCH1
Protein A fragment: 965-974
Protein A site: 14-3-3_pSer970
Protein A modification: (SEP)970
Protein A sequence: QARPVSMPVD
Protein A construct: QARPV(pSer)MPVD (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: 0.6
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): 4.54
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: 4.11
Experiment series 4
Protein A protein: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: SKSFV(pThr)TGIR (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: 0.3
Holdup BI standard deviation: 0.04
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): 4.26
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: 3.57
Experiment series 5
Protein A protein: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: SKSFV(pThr)TGIR (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: 0.36
Holdup BI standard deviation: 0.07
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): 4.03
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: 3.6
Experiment series 6
Protein A protein: PLCH1
Protein A fragment: 1411-1420
Protein A site: 14-3-3_pSer1416
Protein A modification: (SEP)1416
Protein A sequence: ETLRPSVPEI
Protein A construct: ETLRP(pSer)VPEI (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: -0.41
Holdup BI standard deviation: 0.16
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): 3.19
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: PLCH1
Protein A fragment: 1411-1420
Protein A site: 14-3-3_pSer1416
Protein A modification: (SEP)1416
Protein A sequence: ETLRPSVPEI
Protein A construct: ETLRP(pSer)VPEI (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: -0.13
Holdup BI standard deviation: 0.15
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): 1.15
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 8
Protein A protein: PLCH1
Protein A fragment: 918-927
Protein A site: 14-3-3_pSer923
Protein A modification: (SEP)923
Protein A sequence: LRRTASAPAK
Protein A construct: LRRTA(pSer)APAK (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: 0.13
Holdup BI standard deviation: 0.14
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): 0.56
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: PLCH1
Protein A fragment: 1315-1324
Protein A site: 14-3-3_pThr1320
Protein A modification: (TPO)1320
Protein A sequence: LPKSPTKGED
Protein A construct: LPKSP(pThr)KGED (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: -0.05
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): 0.49
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 10
Protein A protein: PLCH1
Protein A fragment: 1315-1324
Protein A site: 14-3-3_pThr1320
Protein A modification: (TPO)1320
Protein A sequence: LPKSPTKGED
Protein A construct: LPKSP(pThr)KGED (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: 0.02
Holdup BI standard deviation: 0.11
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): 0.16
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: PLCH1
Protein A fragment: 918-927
Protein A site: 14-3-3_pSer923
Protein A modification: (SEP)923
Protein A sequence: LRRTASAPAK
Protein A construct: LRRTA(pSer)APAK (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: 0.2
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): 0.05
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: PLCH1
Protein A fragment: 1515-1524
Protein A site: 14-3-3_pThr1520
Protein A modification: (TPO)1520
Protein A sequence: SKSFVTTGIR
Protein A construct: biotin-ttds-SKSFV(pThr)TGIR (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): 380
Standard deviation of dissociation constant (10-6M): 146
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: 3.42