Details of experiments between the fragments P61981~1-247 and Q6ICG6~284-293
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: KIAA0930
Protein B fragment: 284-293
Protein B site: 14-3-3_pSer289
Protein B modification: (SEP)289
Protein B sequence: RVTSFSTPPT
Protein B construct: RVTSF(pSer)TPPT (crude synthetic peptide)
Average holdup BI: 0.95
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): 9.86
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: 5.17
Experiment series 2
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: KIAA0930
Protein B fragment: 284-293
Protein B site: 14-3-3_pSer289
Protein B modification: (SEP)289
Protein B sequence: RVTSFSTPPT
Protein B construct: RVTSF(pSer)TPPT (crude synthetic peptide)
Average holdup BI: 0.96
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): 9.83
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: 5.23
Experiment series 3
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: KIAA0930
Protein B fragment: 284-293
Protein B site: 14-3-3_pSer289
Protein B modification: (SEP)289
Protein B sequence: RVTSFSTPPT
Protein B construct: RVTSF(pSer)TPPT (crude synthetic peptide)
Average holdup BI: 0.97
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 115
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 9.01
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: 5.39
Experiment series 4
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: KIAA0930
Protein B fragment: 284-293
Protein B site: 14-3-3_pSer289
Protein B modification: (SEP)289
Protein B sequence: RVTSFSTPPT
Protein B construct: biotin-ttds-RVTSF(pSer)TPPT (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 6.3
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: 2022.01.18
Number of measurements: 3
Measured pKd: 5.2
Experiment series 5
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: KIAA0930
Protein B fragment: 284-293
Protein B site: 14-3-3_pSer289
Protein B modification: (SEP)289
Protein B sequence: RVTSFSTPPT
Protein B construct: biotin-ttds-RVTSF(pSer)TPPT (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 6.2
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: 2022.01.10
Number of measurements: 3
Measured pKd: 5.21