Details of experiments between the fragments P40818~712-724 and P31946~1-246
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: YWHAB
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3beta
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 134
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: 1
Measured pKd: 5
Experiment series 2
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: YWHAB
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3beta
Average holdup BI: 0.96
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 9.84
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: 5.4