Details of experiments between P63104 and Q6ICG6
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: 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: 0.92
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): 9.43
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: 4.91
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: KIAA0930
Protein B fragment: 257-266
Protein B site: 14-3-3_pSer262
Protein B modification: (SEP)262
Protein B sequence: AVSRVSTGDT
Protein B construct: AVSRV(pSer)TGDT (crude synthetic peptide)
Average holdup BI: 0.06
Holdup BI standard deviation: 0.04
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): 1.71
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: KIAA0930
Protein B fragment: 301-310
Protein B site: 14-3-3_pSer306
Protein B modification: (SEP)306
Protein B sequence: AFFSPSLKRK
Protein B construct: AFFSP(pSer)LKRK (crude synthetic peptide)
Average holdup BI: 0.12
Holdup BI standard deviation: 0.17
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): 0.66
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 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: KIAA0930
Protein B fragment: 399-408
Protein B site: 14-3-3_pThr404
Protein B modification: (TPO)404
Protein B sequence: KPILMT----
Protein B construct: EVRQKPILM(pThr) (crude synthetic peptide)
Average holdup BI: 0.05
Holdup BI standard deviation: 0.23
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): 0.17
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 5
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: 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): 25
Standard deviation of dissociation constant (10-6M): 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: 4.59
Experiment series 6
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: 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): 21
Standard deviation of dissociation constant (10-6M): 2
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: 4.69