Details of experiments between P61981 and O00750
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: PIK3C2B
Protein B fragment: 8-17
Protein B site: 14-3-3_pSer13
Protein B modification: (SEP)13
Protein B sequence: GEHWKSLESV
Protein B construct: GEHWK(pSer)LESV (crude synthetic peptide)
Measured dissociation constant (10-6M): <100
Immobilized partner concentration in holdup experiment (10-6M): 115
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 1
Measured pKd: 4
Experiment series 2
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: PIK3C2B
Protein B fragment: 64-73
Protein B site: 14-3-3_pSer69
Protein B modification: (SEP)69
Protein B sequence: GVDFYSKPAG
Protein B construct: GVDFY(pSer)KPAG (crude synthetic peptide)
Average holdup BI: 0.3
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 115
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.2
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
Measured pKd: 3.59
Experiment series 3
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: PIK3C2B
Protein B fragment: 64-73
Protein B site: 14-3-3_pSer69
Protein B modification: (SEP)69
Protein B sequence: GVDFYSKPAG
Protein B construct: GVDFY(pSer)KPAG (crude synthetic peptide)
Average holdup BI: 0.42
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.17
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
Measured pKd: 3.73
Experiment series 4
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: PIK3C2B
Protein B fragment: 64-73
Protein B site: 14-3-3_pSer69
Protein B modification: (SEP)69
Protein B sequence: GVDFYSKPAG
Protein B construct: GVDFY(pSer)KPAG (crude synthetic peptide)
Average holdup BI: 0.42
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.26
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: 3.75
Experiment series 5
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: PIK3C2B
Protein B fragment: 8-17
Protein B site: 14-3-3_pSer13
Protein B modification: (SEP)13
Protein B sequence: GEHWKSLESV
Protein B construct: GEHWK(pSer)LESV (crude synthetic peptide)
Average holdup BI: 0.5
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.18
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: 3.88