Details of experiments between the fragments P31947~1-248 and Q9Y3M2~2-11
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 modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: CBY1
Protein B fragment: 2-11
Protein B site: 14-3-3_pThr7
Protein B modification: (TPO)7
Protein B sequence: PFFGNTFSPK
Protein B construct: PFFGN(pThr)FSPK (crude synthetic peptide)
Average holdup BI: 0.35
Holdup BI standard deviation: 0.03
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): 5.4
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: 3.68
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: CBY1
Protein B fragment: 2-11
Protein B site: 14-3-3_pThr7
Protein B modification: (TPO)7
Protein B sequence: PFFGNTFSPK
Protein B construct: PFFGN(pThr)FSPK (crude synthetic peptide)
Average holdup BI: 0.39
Holdup BI standard deviation: 0.09
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): 3.94
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: 3.64
Experiment series 3
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: CBY1
Protein B fragment: 2-11
Protein B site: 14-3-3_pThr7
Protein B modification: (TPO)7
Protein B sequence: PFFGNTFSPK
Protein B construct: PFFGN(pThr)FSPK (crude synthetic peptide)
Average holdup BI: 0.33
Holdup BI standard deviation: 0.11
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): 2.37
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: 3.65