Details of experiments between the fragments P31947~1-248 and O60292~1377-1386
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: SIPA1L3
Protein B fragment: 1377-1386
Protein B site: 14-3-3_pSer1382
Protein B modification: (SEP)1382
Protein B sequence: RPKLYSSGSS
Protein B construct: RPKLY(pSer)SGSS (crude synthetic peptide)
Average holdup BI: 0.12
Holdup BI standard deviation: 0.04
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): 3.07
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
The affinity was below the detection threshold of the assay.
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: SIPA1L3
Protein B fragment: 1377-1386
Protein B site: 14-3-3_pSer1382
Protein B modification: (SEP)1382
Protein B sequence: RPKLYSSGSS
Protein B construct: RPKLY(pSer)SGSS (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.05
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): 1.69
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
The affinity was below the detection threshold of the assay.
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: SIPA1L3
Protein B fragment: 1377-1386
Protein B site: 14-3-3_pSer1382
Protein B modification: (SEP)1382
Protein B sequence: RPKLYSSGSS
Protein B construct: RPKLY(pSer)SGSS (crude synthetic peptide)
Average holdup BI: 0.09
Holdup BI standard deviation: 0.07
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): 1.31
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
The affinity was below the detection threshold of the assay.
Experiment series 4
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: SIPA1L3
Protein B fragment: 1377-1386
Protein B site: 14-3-3_pSer1382
Protein B modification: (SEP)1382
Protein B sequence: RPKLYSSGSS
Protein B construct: biotin-ttds-RPKLY(pSer)SGSS (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 821
Standard deviation of dissociation constant (10-6M): 193
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.09
Number of measurements: 3
Measured pKd: 3.09