Details of experiments between P40818 and Q04917
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: USP8
Protein A fragment: 712-724
Protein A site: 14-3-3_pSer718
Protein A modification: (SEP)718
Protein A sequence: LKRSYSSPDI
Protein A construct: KLKRSY(pSer)SPDITQ (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.97
Holdup BI standard deviation: 0.02
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): 9.55
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: 5.42
Experiment series 2
Protein A protein: USP8
Protein A fragment: 713-722
Protein A site: 14-3-3_pSer718
Protein A modification: (SEP)718
Protein A sequence: LKRSYSSPDI
Protein A construct: LKRSY(pSer)SPDI (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.77
Holdup BI standard deviation: 0.03
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): 8.96
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: 4.4
Experiment series 3
Protein A protein: USP8
Protein A fragment: 636-645
Protein A site: 14-3-3_pSer641
Protein A modification: (SEP)641
Protein A sequence: LTRARSEEMG
Protein A construct: LTRAR(pSer)EEMG (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.55
Holdup BI standard deviation: 0.04
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): 6.81
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: 3.98
Experiment series 4
Protein A protein: USP8
Protein A fragment: 686-695
Protein A site: 14-3-3_pSer691
Protein A modification: (SEP)691
Protein A sequence: SSAPPSTPPT
Protein A construct: SSAPP(pSer)TPPT (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: -0.11
Holdup BI standard deviation: 0.11
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): 1.38
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.