Details of experiments between P63104 and Q9P2M7
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: CGN
Protein B fragment: 144-153
Protein B site: 14-3-3_pSer149
Protein B modification: (SEP)149
Protein B sequence: SNRSNSMLEL
Protein B construct: SNRSN(pSer)MLEL (crude synthetic peptide)
Average holdup BI: 0.38
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.09
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
Measured pKd: 3.67
Experiment series 2
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: CGN
Protein B fragment: 126-135
Protein B site: 14-3-3_pSer131
Protein B modification: (SEP)131
Protein B sequence: LLRSHSQASL
Protein B construct: LLRSH(pSer)QASL (crude synthetic peptide)
Average holdup BI: 0.24
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.74
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: 3.37
Experiment series 3
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: CGN
Protein B fragment: 66-75
Protein B site: 14-3-3_pSer71
Protein B modification: (SEP)71
Protein B sequence: EKGGDSFGVQ
Protein B construct: EKGGD(pSer)FGVQ (crude synthetic peptide)
Average holdup BI: -0.04
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.3
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 4
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: CGN
Protein B fragment: 327-336
Protein B site: 14-3-3_pSer332
Protein B modification: (SEP)332
Protein B sequence: VRRKVSLVLE
Protein B construct: VRRKV(pSer)LVLE (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.21
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.04
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: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: CGN
Protein B fragment: 126-135
Protein B site: 14-3-3_pSer131
Protein B modification: (SEP)131
Protein B sequence: LLRSHSQASL
Protein B construct: biotin-ttds-LLRSH(pSer)QASL (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 105
Standard deviation of dissociation constant (10-6M): 2
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: 3.98
Experiment series 6
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: CGN
Protein B fragment: 126-135
Protein B site: 14-3-3_pSer131
Protein B modification: (SEP)131
Protein B sequence: LLRSHSQASL
Protein B construct: biotin-ttds-LLRSH(pSer)QASL (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 96
Standard deviation of dissociation constant (10-6M): 30
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.10
Number of measurements: 3
Measured pKd: 4.02