Details of experiments between Q04917 and P33176
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: KIF5B
Protein B fragment: 945-954
Protein B site: 14-3-3_pSer950
Protein B modification: (SEP)950
Protein B sequence: AFVQNSQPVA
Protein B construct: AFVQN(pSer)QPVA (crude synthetic peptide)
Average holdup BI: 0.13
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.69
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: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: KIF5B
Protein B fragment: 446-455
Protein B site: 14-3-3_pThr451
Protein B modification: (TPO)451
Protein B sequence: VEKLKTQMLD
Protein B construct: VEKLK(pThr)QMLD (crude synthetic peptide)
Average holdup BI: 0.44
Holdup BI standard deviation: 0.35
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.36
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: 3.78
Experiment series 3
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: KIF5B
Protein B fragment: 149-158
Protein B site: 14-3-3_pSer154
Protein B modification: (SEP)154
Protein B sequence: SKTNLSVHED
Protein B construct: SKTNL(pSer)VHED (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.23
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 4
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: KIF5B
Protein B fragment: 392-401
Protein B site: 14-3-3_pThr397
Protein B modification: (TPO)397
Protein B sequence: VDKDITLTND
Protein B construct: VDKDI(pThr)LTND (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.1
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.1
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
The affinity was below the detection threshold of the assay.
Experiment series 5
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: KIF5B
Protein B fragment: 933-942
Protein B site: 14-3-3_pSer938
Protein B modification: (SEP)938
Protein B sequence: SPTHPSAIRG
Protein B construct: SPTHP(pSer)AIRG (crude synthetic peptide)
Average holdup BI: 0
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.09
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.