Details of experiments between P27348 and O96013
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: PAK4
Protein B fragment: 202-211
Protein B site: 14-3-3_pThr207
Protein B modification: (TPO)207
Protein B sequence: GRPFNTYPRA
Protein B construct: GRPFN(pThr)YPRA (crude synthetic peptide)
Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 130
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: 1
Measured pKd: 5
Experiment series 2
Protein A protein: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: PAK4
Protein B fragment: 262-271
Protein B site: 14-3-3_pSer267
Protein B modification: (SEP)267
Protein B sequence: LGPHASEPQL
Protein B construct: LGPHA(pSer)EPQL (crude synthetic peptide)
Average holdup BI: 0.37
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): 5.58
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: 3.66
Experiment series 3
Protein A protein: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: PAK4
Protein B fragment: 176-185
Protein B site: 14-3-3_pSer181
Protein B modification: (SEP)181
Protein B sequence: DKRPLSGPDV
Protein B construct: DKRPL(pSer)GPDV (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.03
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): 3.15
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: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: PAK4
Protein B fragment: 469-478
Protein B site: 14-3-3_pSer474
Protein B modification: (SEP)474
Protein B sequence: VPRRKSLVGT
Protein B construct: VPRRK(pSer)LVGT (crude synthetic peptide)
Average holdup BI: 0.07
Holdup BI standard deviation: 0.11
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): 0.65
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: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: PAK4
Protein B fragment: 162-171
Protein B site: 14-3-3_pSer167
Protein B modification: (SEP)167
Protein B sequence: SSREGSGGPQ
Protein B construct: SSREG(pSer)GGPQ (crude synthetic peptide)
Average holdup BI: 0.09
Holdup BI standard deviation: 0.65
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.