Details of experiments between the fragments P30305~146-155 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: CDC25B
Protein A fragment: 146-155
Protein A site: 14-3-3_pSer151
Protein A modification: (SEP)151
Protein A sequence: IRRFQSMPVR
Protein A construct: IRRFQ(pSer)MPVR (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
Average holdup BI: 0.91
Holdup BI standard deviation: 0.02
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): 8.1
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: 4.84
Experiment series 2
Protein A protein: CDC25B
Protein A fragment: 146-155
Protein A site: 14-3-3_pSer151
Protein A modification: (SEP)151
Protein A sequence: IRRFQSMPDG
Protein A construct: IRRFQ(pSer)MPDG (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
Average holdup BI: 0.7
Holdup BI standard deviation: 0.02
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): 7.71
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: 4.2
Experiment series 3
Protein A protein: CDC25B
Protein A fragment: 146-155
Protein A site: 14-3-3_pSer151
Protein A modification: (SEP)151
Protein A sequence: IRRFQSMPDG
Protein A construct: IRRFQ(pSer)MPDG (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
Average holdup BI: 0.65
Holdup BI standard deviation: 0.06
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): 4.94
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: 4.21
Experiment series 4
Protein A protein: CDC25B
Protein A fragment: 146-155
Protein A site: 14-3-3_pSer151
Protein A modification: (SEP)151
Protein A sequence: IRRFQSMPVR
Protein A construct: IRRFQ(pSer)MPVR (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
Average holdup BI: 0.79
Holdup BI standard deviation: 0.06
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): 4.67
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: 4.53
Experiment series 5
Protein A protein: CDC25B
Protein A fragment: 146-155
Protein A site: 14-3-3_pSer151
Protein A modification: (SEP)151
Protein A sequence: IRRFQSMPVR
Protein A construct: IRRFQ(pSer)MPVR (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: 0.97
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): 3.28
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: 5.44
Experiment series 6
Protein A protein: CDC25B
Protein A fragment: 146-155
Protein A site: 14-3-3_pSer151
Protein A modification: (SEP)151
Protein A sequence: IRRFQSMPDG
Protein A construct: IRRFQ(pSer)MPDG (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: 0.06
Holdup BI standard deviation: 0.1
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): 0.52
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.