Details of experiments between P62258 and Q9Y3M2
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: CBY1
Protein B fragment: 15-24
Protein B site: 14-3-3_pSer20
Protein B modification: (SEP)20
Protein B sequence: PRKSASLSNL
Protein B construct: PRKSA(pSer)LSNL (crude synthetic peptide)
Average holdup BI: 0.72
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): 7.94
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: 4.34
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: CBY1
Protein B fragment: 15-24
Protein B site: 14-3-3_pSer20
Protein B modification: (SEP)20
Protein B sequence: PRKSASLSNL
Protein B construct: PRKSA(pSer)LSNL (crude synthetic peptide)
Average holdup BI: 0.77
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): 7.22
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: 4.37
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: CBY1
Protein B fragment: 2-11
Protein B site: 14-3-3_pThr7
Protein B modification: (TPO)7
Protein B sequence: PFFGNTFSPK
Protein B construct: PFFGN(pThr)FSPK (crude synthetic peptide)
Average holdup BI: 0.32
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): 4.19
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: 3.53
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: CBY1
Protein B fragment: 2-11
Protein B site: 14-3-3_pThr7
Protein B modification: (TPO)7
Protein B sequence: PFFGNTFSPK
Protein B construct: PFFGN(pThr)FSPK (crude synthetic peptide)
Average holdup BI: 0.23
Holdup BI standard deviation: 0.13
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): 1.56
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 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: CBY1
Protein B fragment: 21-30
Protein B site: 14-3-3_pSer26
Protein B modification: (SEP)26
Protein B sequence: LSNLHSLDRS
Protein B construct: LSNLH(pSer)LDRS (crude synthetic peptide)
Average holdup BI: -0.06
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): 1.06
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.