Details of experiments between the fragments P46937~271-280 and P31947~1-248
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YAP1
Protein A fragment: 271-280
Protein A site: 14-3-3_pSer276
Protein A modification: (SEP)276
Protein A sequence: QRISQSAPVK
Protein A construct: QRISQ(pSer)APVK (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Average holdup BI: 0.99
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 10.88
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: 5.69
Experiment series 2
Protein A protein: YAP1
Protein A fragment: 271-280
Protein A site: 14-3-3_pSer276
Protein A modification: (SEP)276
Protein A sequence: QRISQSAPVK
Protein A construct: QRISQ(pSer)APVK (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Average holdup BI: 0.99
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.19
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: 5.76
Experiment series 3
Protein A protein: YAP1
Protein A fragment: 271-280
Protein A site: 14-3-3_pSer276
Protein A modification: (SEP)276
Protein A sequence: QRISQSAPVK
Protein A construct: QRISQ(pSer)APVK (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Average holdup BI: 0.99
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.31
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: 5.79
Experiment series 4
Protein A protein: YAP1
Protein A fragment: 271-280
Protein A site: 14-3-3_pSer276
Protein A modification: (SEP)276
Protein A sequence: QRISQSAPVK
Protein A construct: biotin-ttds-QRISQ(pSer)APVK (HPLC-purified synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Measured dissociation constant (10-6M): 3.7
Standard deviation of dissociation constant (10-6M): 0.7
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: 5.43