Details of experiments between P61981 and Q9UQL6
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: HDAC5
Protein B fragment: 493-502
Protein B site: 14-3-3_pSer498
Protein B modification: (SEP)498
Protein B sequence: LSRTQSSPLP
Protein B construct: LSRTQ(pSer)SPLP (crude synthetic peptide)
Measured dissociation constant (10-6M): <10
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: 5
Experiment series 2
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: HDAC5
Protein B fragment: 254-263
Protein B site: 14-3-3_pSer259
Protein B modification: (SEP)259
Protein B sequence: LRKTASEPNL
Protein B construct: LRKTA(pSer)EPNL (crude synthetic peptide)
Average holdup BI: 0.95
Holdup BI standard deviation: 0.01
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): 9.32
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: 5.16
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: HDAC5
Protein B fragment: 254-263
Protein B site: 14-3-3_pSer259
Protein B modification: (SEP)259
Protein B sequence: LRKTASEPNL
Protein B construct: LRKTA(pSer)EPNL (crude synthetic peptide)
Average holdup BI: 0.94
Holdup BI standard deviation: 0.02
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): 8.22
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: 5.05
Experiment series 4
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: HDAC5
Protein B fragment: 254-263
Protein B site: 14-3-3_pSer259
Protein B modification: (SEP)259
Protein B sequence: LRKTASEPNL
Protein B construct: LRKTA(pSer)EPNL (crude synthetic peptide)
Average holdup BI: 0.92
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): 6.64
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: 5.03
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: HDAC5
Protein B fragment: 493-502
Protein B site: 14-3-3_pSer498
Protein B modification: (SEP)498
Protein B sequence: LSRTQSSPLP
Protein B construct: LSRTQ(pSer)SPLP (crude synthetic peptide)
Average holdup BI: 0.8
Holdup BI standard deviation: 0.13
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): 3.07
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: 4.47
Experiment series 6
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: HDAC5
Protein B fragment: 542-551
Protein B site: 14-3-3_pThr547
Protein B modification: (TPO)547
Protein B sequence: PRQPTTHPEE
Protein B construct: PRQPT(pThr)HPEE (crude synthetic peptide)
Average holdup BI: 0.32
Holdup BI standard deviation: 0.13
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): 2.63
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: 3.55
Experiment series 7
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: HDAC5
Protein B fragment: 542-551
Protein B site: 14-3-3_pThr547
Protein B modification: (TPO)547
Protein B sequence: PRQPTTHPEE
Protein B construct: PRQPT(pThr)HPEE (crude synthetic peptide)
Average holdup BI: 0.23
Holdup BI standard deviation: 0.13
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): 1.65
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
The affinity was below the detection threshold of the assay.
Experiment series 8
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: HDAC5
Protein B fragment: 542-551
Protein B site: 14-3-3_pThr547
Protein B modification: (TPO)547
Protein B sequence: PRQPTTHPEE
Protein B construct: PRQPT(pThr)HPEE (crude synthetic peptide)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.24
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): 0.38
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
The affinity was below the detection threshold of the assay.