Details of experiments between the fragments Q14153~164-173 and P61981~1-247
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: FAM53B
Protein A fragment: 164-173
Protein A site: 14-3-3_pSer169
Protein A modification: (SEP)169
Protein A sequence: RSSSFSLPSR
Protein A construct: RSSSF(pSer)LPSR (crude synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): <1
Immobilized partner concentration in holdup experiment (10-6M): 115
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: 6
Experiment series 2
Protein A protein: FAM53B
Protein A fragment: 164-173
Protein A site: 14-3-3_pSer169
Protein A modification: (SEP)169
Protein A sequence: RSSSFSLPSR
Protein A construct: RSSSF(pSer)LPSR (crude synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Average holdup BI: 0.99
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.76
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: 5.82
Experiment series 3
Protein A protein: FAM53B
Protein A fragment: 164-173
Protein A site: 14-3-3_pSer169
Protein A modification: (SEP)169
Protein A sequence: RSSSFSLPSR
Protein A construct: RSSSF(pSer)LPSR (crude synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Average holdup BI: 0.99
Holdup BI standard deviation: 0
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): 6.65
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: 5.97
Experiment series 4
Protein A protein: FAM53B
Protein A fragment: 164-173
Protein A site: 14-3-3_pSer169
Protein A modification: (SEP)169
Protein A sequence: RSSSFSLPSR
Protein A construct: biotin-ttds-RSSSF(pSer)LPSR (HPLC-purified synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): 5.8
Standard deviation of dissociation constant (10-6M): 1
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.01.18
Number of measurements: 3
Measured pKd: 5.23
Experiment series 5
Protein A protein: FAM53B
Protein A fragment: 164-173
Protein A site: 14-3-3_pSer169
Protein A modification: (SEP)169
Protein A sequence: RSSSFSLPSR
Protein A construct: biotin-ttds-RSSSF(pSer)LPSR (HPLC-purified synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Measured dissociation constant (10-6M): 3.4
Standard deviation of dissociation constant (10-6M): 0.5
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.01.10
Number of measurements: 3
Measured pKd: 5.46