Details of experiments between Q04917 and Q9H0B6
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: KLC2
Protein B fragment: 577-586
Protein B site: 14-3-3_pSer582
Protein B modification: (SEP)582
Protein B sequence: MKRASSLNFL
Protein B construct: MKRAS(pSer)LNFL (crude synthetic peptide)
Average holdup BI: 0.89
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): 6.31
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: 4.78
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: KLC2
Protein B fragment: 605-614
Protein B site: 14-3-3_pSer610
Protein B modification: (SEP)610
Protein B sequence: RTLSSSSMDL
Protein B construct: RTLSS(pSer)SMDL (crude synthetic peptide)
Average holdup BI: 0.45
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): 5.34
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: 3.79
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: KLC2
Protein B fragment: 614-623
Protein B site: 14-3-3_pSer619
Protein B modification: (SEP)619
Protein B sequence: LSRRSSLVG-
Protein B construct: DLSRRS(pSer)LVG (crude synthetic peptide)
Average holdup BI: 0.22
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): 3.95
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: 3.33
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: KLC2
Protein B fragment: 603-612
Protein B site: 14-3-3_pSer608
Protein B modification: (SEP)608
Protein B sequence: DSRTLSSSSM
Protein B construct: DSRTL(pSer)SSSM (crude synthetic peptide)
Average holdup BI: 0.21
Holdup BI standard deviation: 0.06
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): 3.86
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: 3.31
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: KLC2
Protein B fragment: 540-549
Protein B site: 14-3-3_pSer545
Protein B modification: (SEP)545
Protein B sequence: LRRSGSFGKL
Protein B construct: LRRSG(pSer)FGKL (crude synthetic peptide)
Average holdup BI: 0.35
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): 3.38
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: 3.61
Experiment series 6
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: KLC2
Protein B fragment: 584-593
Protein B site: 14-3-3_pSer589
Protein B modification: (SEP)589
Protein B sequence: NFLNKSVEEP
Protein B construct: NFLNK(pSer)VEEP (crude synthetic peptide)
Average holdup BI: 0.63
Holdup BI standard deviation: 0.09
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): 3.18
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: 4.11
Experiment series 7
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: KLC2
Protein B fragment: 610-619
Protein B site: 14-3-3_pSer615
Protein B modification: (SEP)615
Protein B sequence: SSMDLSRRSS
Protein B construct: SSMDL(pSer)RRSS (crude synthetic peptide)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.06
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): 0.64
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.