Details of experiments between P63104 and Q14153
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FAM53B
Protein B fragment: 164-173
Protein B site: 14-3-3_pSer169
Protein B modification: (SEP)169
Protein B sequence: RSSSFSLPSR
Protein B construct: RSSSF(pSer)LPSR (crude synthetic peptide)
Average holdup BI: 0.96
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.68
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: 5.27
Experiment series 2
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FAM53B
Protein B fragment: 263-272
Protein B site: 14-3-3_pSer268
Protein B modification: (SEP)268
Protein B sequence: LSRSRSQPCV
Protein B construct: LSRSR(pSer)QPCV (crude synthetic peptide)
Average holdup BI: -0.23
Holdup BI standard deviation: 0.56
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.45
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 3
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FAM53B
Protein B fragment: 164-173
Protein B site: 14-3-3_pSer169
Protein B modification: (SEP)169
Protein B sequence: RSSSFSLPSR
Protein B construct: biotin-ttds-RSSSF(pSer)LPSR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 14
Standard deviation of dissociation constant (10-6M): 1.2
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.01.19
Number of measurements: 3
Measured pKd: 4.85
Experiment series 4
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: FAM53B
Protein B fragment: 164-173
Protein B site: 14-3-3_pSer169
Protein B modification: (SEP)169
Protein B sequence: RSSSFSLPSR
Protein B construct: biotin-ttds-RSSSF(pSer)LPSR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 12
Standard deviation of dissociation constant (10-6M): 0.9
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.10
Number of measurements: 3
Measured pKd: 4.93