Details of experiments between P31946 and Q92934
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: BAD
Protein B fragment: 129-138
Protein B site: 14-3-3_pSer134
Protein B modification: (SEP)134
Protein B sequence: LPRPKSAGTA
Protein B construct: LPRPK(pSer)AGTA (crude synthetic peptide)
Average holdup BI: 0.59
Holdup BI standard deviation: 0.07
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): 5.87
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: 4.14
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: BAD
Protein B fragment: 94-103
Protein B site: 14-3-3_pSer99
Protein B modification: (SEP)99
Protein B sequence: RGRSRSAPPN
Protein B construct: RGRSR(pSer)APPN (crude synthetic peptide)
Average holdup BI: 0.77
Holdup BI standard deviation: 0.11
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): 5.07
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: 4.39
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: BAD
Protein B fragment: 129-138
Protein B site: 14-3-3_pSer134
Protein B modification: (SEP)134
Protein B sequence: LPRPKSAGTA
Protein B construct: LPRPK(pSer)AGTA (crude synthetic peptide)
Average holdup BI: 0.57
Holdup BI standard deviation: 0.04
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): 5.07
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
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: BAD
Protein B fragment: 113-122
Protein B site: 14-3-3_pSer118
Protein B modification: (SEP)118
Protein B sequence: ELRRMSDEFV
Protein B construct: ELRRM(pSer)DEFV (crude synthetic peptide)
Average holdup BI: 0.46
Holdup BI standard deviation: 0.15
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): 2.88
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: 3.8
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: BAD
Protein B fragment: 119-128
Protein B site: 14-3-3_pSer124
Protein B modification: (SEP)124
Protein B sequence: DEFVDSFKKG
Protein B construct: DEFVD(pSer)FKKG (crude synthetic peptide)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.02
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): 2.14
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 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: BAD
Protein B fragment: 113-122
Protein B site: 14-3-3_pSer118
Protein B modification: (SEP)118
Protein B sequence: ELRRMSDEFV
Protein B construct: ELRRM(pSer)DEFV (crude synthetic peptide)
Average holdup BI: 0.07
Holdup BI standard deviation: 0.08
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): 0.94
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 7
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: BAD
Protein B fragment: 66-75
Protein B site: 14-3-3_pSer71
Protein B modification: (SEP)71
Protein B sequence: AVEIRSRHSS
Protein B construct: AVEIR(pSer)RHSS (crude synthetic peptide)
Average holdup BI: 0.1
Holdup BI standard deviation: 0.34
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): 0.22
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 8
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: BAD
Protein B fragment: 119-128
Protein B site: 14-3-3_pSer124
Protein B modification: (SEP)124
Protein B sequence: DEFVDSFKKG
Protein B construct: DEFVD(pSer)FKKG (crude synthetic peptide)
Average holdup BI: -0.01
Holdup BI standard deviation: 0.04
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): 0.22
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
The affinity was below the detection threshold of the assay.