Details of experiments between the fragments P62258~1-255 and P46937~271-280
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: YAP1
Protein B fragment: 271-280
Protein B site: 14-3-3_pSer276
Protein B modification: (SEP)276
Protein B sequence: QRISQSAPVK
Protein B construct: QRISQ(pSer)APVK (crude synthetic peptide)
Average holdup BI: 0.98
Holdup BI standard deviation: 0
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): 11.08
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: 5.58
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: YAP1
Protein B fragment: 271-280
Protein B site: 14-3-3_pSer276
Protein B modification: (SEP)276
Protein B sequence: QRISQSAPVK
Protein B construct: QRISQ(pSer)APVK (crude synthetic peptide)
Average holdup BI: 0.98
Holdup BI standard deviation: 0
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): 6.16
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: 5.64
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: YAP1
Protein B fragment: 271-280
Protein B site: 14-3-3_pSer276
Protein B modification: (SEP)276
Protein B sequence: QRISQSAPVK
Protein B construct: biotin-ttds-QRISQ(pSer)APVK (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 2.8
Standard deviation of dissociation constant (10-6M): 0.4
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: 5.55