Details of experiments between Q6ICG6 and P27348
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: KIAA0930
Protein A fragment: 284-293
Protein A site: 14-3-3_pSer289
Protein A modification: (SEP)289
Protein A sequence: RVTSFSTPPT
Protein A construct: RVTSF(pSer)TPPT (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.91
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 9.33
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: 4.89
Experiment series 2
Protein A protein: KIAA0930
Protein A fragment: 301-310
Protein A site: 14-3-3_pSer306
Protein A modification: (SEP)306
Protein A sequence: AFFSPSLKRK
Protein A construct: AFFSP(pSer)LKRK (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: -0.15
Holdup BI standard deviation: 0.23
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.62
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: KIAA0930
Protein A fragment: 399-408
Protein A site: 14-3-3_pThr404
Protein A modification: (TPO)404
Protein A sequence: KPILMT----
Protein A construct: EVRQKPILM(pThr) (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: -0.19
Holdup BI standard deviation: 0.35
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.57
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: KIAA0930
Protein A fragment: 257-266
Protein A site: 14-3-3_pSer262
Protein A modification: (SEP)262
Protein A sequence: AVSRVSTGDT
Protein A construct: AVSRV(pSer)TGDT (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.02
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.47
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: KIAA0930
Protein A fragment: 284-293
Protein A site: 14-3-3_pSer289
Protein A modification: (SEP)289
Protein A sequence: RVTSFSTPPT
Protein A construct: biotin-ttds-RVTSF(pSer)TPPT (HPLC-purified synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Measured dissociation constant (10-6M): 19
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: 2022.02.08
Number of measurements: 3
Measured pKd: 4.72