Details of experiments between the fragments O96013~202-211 and P31947~1-248
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: PAK4
Protein A fragment: 202-211
Protein A site: 14-3-3_pThr207
Protein A modification: (TPO)207
Protein A sequence: GRPFNTYPRA
Protein A construct: GRPFN(pThr)YPRA (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 113
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: PAK4
Protein A fragment: 202-211
Protein A site: 14-3-3_pThr207
Protein A modification: (TPO)207
Protein A sequence: GRPFNTYPRA
Protein A construct: GRPFN(pThr)YPRA (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 142
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: 1
Measured pKd: 5
Experiment series 3
Protein A protein: PAK4
Protein A fragment: 202-211
Protein A site: 14-3-3_pThr207
Protein A modification: (TPO)207
Protein A sequence: GRPFNTYPRA
Protein A construct: GRPFN(pThr)YPRA (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Average holdup BI: 0.93
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.55
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: 4
Measured pKd: 5.08