Details of experiments between P63104 and P40818
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: USP8
Protein B fragment: 712-724
Protein B site: 14-3-3_pSer718
Protein B modification: (SEP)718
Protein B sequence: LKRSYSSPDI
Protein B construct: KLKRSY(pSer)SPDITQ (crude synthetic peptide)
Average holdup BI: 0.96
Holdup BI standard deviation: 0.01
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): 9.88
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: 5.29
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: USP8
Protein B fragment: 713-722
Protein B site: 14-3-3_pSer718
Protein B modification: (SEP)718
Protein B sequence: LKRSYSSPDI
Protein B construct: LKRSY(pSer)SPDI (crude synthetic peptide)
Average holdup BI: 0.76
Holdup BI standard deviation: 0.05
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): 7.62
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
Measured pKd: 4.39
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: USP8
Protein B fragment: 636-645
Protein B site: 14-3-3_pSer641
Protein B modification: (SEP)641
Protein B sequence: LTRARSEEMG
Protein B construct: LTRAR(pSer)EEMG (crude synthetic peptide)
Average holdup BI: 0.44
Holdup BI standard deviation: 0.05
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): 5.98
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: 3.77
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: USP8
Protein B fragment: 686-695
Protein B site: 14-3-3_pSer691
Protein B modification: (SEP)691
Protein B sequence: SSAPPSTPPT
Protein B construct: SSAPP(pSer)TPPT (crude synthetic peptide)
Average holdup BI: -0.04
Holdup BI standard deviation: 0.11
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): 0.4
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.