Details of experiments between the fragments P31947~1-248 and Q92974~117-126
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: ARHGEF2
Protein B fragment: 117-126
Protein B site: 14-3-3_pSer122
Protein B modification: (SEP)122
Protein B sequence: RERPSSAIYP
Protein B construct: RERPS(pSer)AIYP (crude synthetic peptide)
Average holdup BI: 0.45
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.47
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
Measured pKd: 3.76
Experiment series 2
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: ARHGEF2
Protein B fragment: 117-126
Protein B site: 14-3-3_pSer122
Protein B modification: (SEP)122
Protein B sequence: RERPSSAIYP
Protein B construct: RERPS(pSer)AIYP (crude synthetic peptide)
Average holdup BI: 0.33
Holdup BI standard deviation: 0.13
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.56
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: 3.63
Experiment series 3
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: ARHGEF2
Protein B fragment: 117-126
Protein B site: 14-3-3_pSer122
Protein B modification: (SEP)122
Protein B sequence: RERPSSAIYP
Protein B construct: RERPS(pSer)AIYP (crude synthetic peptide)
Average holdup BI: 0.32
Holdup BI standard deviation: 0.2
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): 1.64
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: 3.62