Details of experiments between Q04917 and Q9UQC2
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: GAB2
Protein B fragment: 128-137
Protein B site: 14-3-3_pSer133
Protein B modification: (SEP)133
Protein B sequence: LRNVSSAGHG
Protein B construct: LRNVS(pSer)AGHG (crude synthetic peptide)
Measured dissociation constant (10-6M): <100
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: 4
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: GAB2
Protein B fragment: 386-395
Protein B site: 14-3-3_pThr391
Protein B modification: (TPO)391
Protein B sequence: IPRRNTLPAM
Protein B construct: IPRRN(pThr)LPAM (crude synthetic peptide)
Average holdup BI: 0.95
Holdup BI standard deviation: 0.02
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): 8.13
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: 5.19
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: GAB2
Protein B fragment: 380-389
Protein B site: 14-3-3_pThr385
Protein B modification: (TPO)385
Protein B sequence: RSVAATIPRR
Protein B construct: RSVAA(pThr)IPRR (crude synthetic peptide)
Average holdup BI: 0.73
Holdup BI standard deviation: 0.07
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): 6.27
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: 4.31
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: GAB2
Protein B fragment: 205-214
Protein B site: 14-3-3_pSer210
Protein B modification: (SEP)210
Protein B sequence: NARSASFSQG
Protein B construct: NARSA(pSer)FSQG (crude synthetic peptide)
Average holdup BI: 0.37
Holdup BI standard deviation: 0.08
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): 4.54
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 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: GAB2
Protein B fragment: 207-216
Protein B site: 14-3-3_pSer212
Protein B modification: (SEP)212
Protein B sequence: RSASFSQGTR
Protein B construct: RSASF(pSer)QGTR (crude synthetic peptide)
Average holdup BI: 0.51
Holdup BI standard deviation: 0.15
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): 3.47
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: 3.89
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: GAB2
Protein B fragment: 317-326
Protein B site: 14-3-3_pSer322
Protein B modification: (SEP)322
Protein B sequence: PATPLSAYQI
Protein B construct: PATPL(pSer)AYQI (crude synthetic peptide)
Average holdup BI: 0.21
Holdup BI standard deviation: 0.12
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): 1.9
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: 3.3
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: GAB2
Protein B fragment: 467-476
Protein B site: 14-3-3_pSer472
Protein B modification: (SEP)472
Protein B sequence: RAGDNSQSVY
Protein B construct: RAGDN(pSer)QSVY (crude synthetic peptide)
Average holdup BI: -0.07
Holdup BI standard deviation: 0.07
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): 1.16
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 8
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: GAB2
Protein B fragment: 259-268
Protein B site: 14-3-3_pSer264
Protein B modification: (SEP)264
Protein B sequence: TEFRDSTYDL
Protein B construct: TEFRD(pSer)TYDL (crude synthetic peptide)
Average holdup BI: 0.05
Holdup BI standard deviation: 0.14
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): 0.3
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 9
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: GAB2
Protein B fragment: 207-216
Protein B site: 14-3-3_pSer212
Protein B modification: (SEP)212
Protein B sequence: RSASFSQGTR
Protein B construct: biotin-ttds-RSASF(pSer)QGTR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 83
Standard deviation of dissociation constant (10-6M): 12
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.01.19
Number of measurements: 3
Measured pKd: 4.08