Details of experiments between the fragments P31947~1-248 and P30305~558-567
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: CDC25B
Protein B fragment: 558-567
Protein B site: 14-3-3_pSer563
Protein B modification: (SEP)563
Protein B sequence: RLKTRSWAGE
Protein B construct: RLKTR(pSer)WAGE (crude synthetic peptide)
Average holdup BI: 0.72
Holdup BI standard deviation: 0.03
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): 7.43
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: 4.25
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: CDC25B
Protein B fragment: 558-567
Protein B site: 14-3-3_pSer563
Protein B modification: (SEP)563
Protein B sequence: RLKTRSWAGE
Protein B construct: RLKTR(pSer)WAGE (crude synthetic peptide)
Average holdup BI: 0.63
Holdup BI standard deviation: 0.03
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): 7.05
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: 4.17
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: CDC25B
Protein B fragment: 558-567
Protein B site: 14-3-3_pSer563
Protein B modification: (SEP)563
Protein B sequence: RLKTRSWAGE
Protein B construct: RLKTR(pSer)WAGE (crude synthetic peptide)
Average holdup BI: 0.62
Holdup BI standard deviation: 0.06
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): 4.22
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: 4.17