Details of experiments between P31946 and Q86TI0
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 232-241
Protein B site: 14-3-3_pSer237
Protein B modification: (SEP)237
Protein B sequence: MRKSFSQPGL
Protein B construct: MRKSF(pSer)QPGL (crude synthetic peptide)
Measured dissociation constant (10-6M): <100
Immobilized partner concentration in holdup experiment (10-6M): 134
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: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 622-631
Protein B site: 14-3-3_pSer627
Protein B modification: (SEP)627
Protein B sequence: LMRYHSVSTE
Protein B construct: LMRYH(pSer)VSTE (crude synthetic peptide)
Average holdup BI: 0.47
Holdup BI standard deviation: 0.1
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): 3.2
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: 3.81
Experiment series 3
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 622-631
Protein B site: 14-3-3_pSer627
Protein B modification: (SEP)627
Protein B sequence: LMRYHSVSTE
Protein B construct: LMRYH(pSer)VSTE (crude synthetic peptide)
Average holdup BI: 0.19
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.68
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: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 232-241
Protein B site: 14-3-3_pSer237
Protein B modification: (SEP)237
Protein B sequence: MRKSFSQPGL
Protein B construct: MRKSF(pSer)QPGL (crude synthetic peptide)
Average holdup BI: 0.65
Holdup BI standard deviation: 0.12
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.55
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: 4.25
Experiment series 5
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 561-570
Protein B site: 14-3-3_pSer566
Protein B modification: (SEP)566
Protein B sequence: KLLGSSEDLS
Protein B construct: KLLGS(pSer)EDLS (crude synthetic peptide)
Average holdup BI: 0.02
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): 1.09
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 6
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 561-570
Protein B site: 14-3-3_pSer566
Protein B modification: (SEP)566
Protein B sequence: KLLGSSEDLS
Protein B construct: KLLGS(pSer)EDLS (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.12
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.65
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 7
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 591-600
Protein B site: 14-3-3_pThr596
Protein B modification: (TPO)596
Protein B sequence: RRRANTLSHF
Protein B construct: RRRAN(pThr)LSHF (crude synthetic peptide)
Average holdup BI: -0.16
Holdup BI standard deviation: 0.68
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.18
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
The affinity was below the detection threshold of the assay.
Experiment series 8
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: TBC1D1
Protein B fragment: 566-575
Protein B site: 14-3-3_pSer571
Protein B modification: (SEP)571
Protein B sequence: SEDLSSDSES
Protein B construct: SEDLS(pSer)DSES (crude synthetic peptide)
Average holdup BI: 0.04
Holdup BI standard deviation: 1.48
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.02
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.