Details of experiments between P31946 and O95685
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: PPP1R3D
Protein B fragment: 69-78
Protein B site: 14-3-3_pSer74
Protein B modification: (SEP)74
Protein B sequence: LRRARSLPSS
Protein B construct: LRRAR(pSer)LPSS (crude synthetic peptide)
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 134
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: 5
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: PPP1R3D
Protein B fragment: 69-78
Protein B site: 14-3-3_pSer74
Protein B modification: (SEP)74
Protein B sequence: LRRARSLPSS
Protein B construct: LRRAR(pSer)LPSS (crude synthetic peptide)
Average holdup BI: 0.99
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): 6.38
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: 5.83
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: PPP1R3D
Protein B fragment: 72-81
Protein B site: 14-3-3_pSer77
Protein B modification: (SEP)77
Protein B sequence: ARSLPSSPER
Protein B construct: ARSLP(pSer)SPER (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.09
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): 1.02
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 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: PPP1R3D
Protein B fragment: 72-81
Protein B site: 14-3-3_pSer77
Protein B modification: (SEP)77
Protein B sequence: ARSLPSSPER
Protein B construct: ARSLP(pSer)SPER (crude synthetic peptide)
Average holdup BI: 0.03
Holdup BI standard deviation: 0.09
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): 0.34
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.