Details of experiments between P31946 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: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
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: 0.85
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 8.19
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
Measured pKd: 4.62
Experiment series 2
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
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: 0.84
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.65
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: 4.72
Experiment series 3
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
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: 0.45
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5
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: 3.9
Experiment series 4
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
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: 0.49
Holdup BI standard deviation: 0.1
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.09
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: 3.85
Experiment series 5
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
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: 0.1
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.67
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: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
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: -0.27
Holdup BI standard deviation: 0.54
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.59
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.