Details of experiments between P31946 and Q8IVT5
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: KSR1
Protein B fragment: 401-410
Protein B site: 14-3-3_pSer406
Protein B modification: (SEP)406
Protein B sequence: LRRTESVPSD
Protein B construct: LRRTE(pSer)VPSD (crude synthetic peptide)
Average holdup BI: 0.48
Holdup BI standard deviation: 0.03
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): 7.46
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: 3.97
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: KSR1
Protein B fragment: 401-410
Protein B site: 14-3-3_pSer406
Protein B modification: (SEP)406
Protein B sequence: LRRTESVPSD
Protein B construct: LRRTE(pSer)VPSD (crude synthetic peptide)
Average holdup BI: 0.49
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): 5.72
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: 3.85
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: KSR1
Protein B fragment: 326-335
Protein B site: 14-3-3_pSer331
Protein B modification: (SEP)331
Protein B sequence: MRFDLSHGSP
Protein B construct: MRFDL(pSer)HGSP (crude synthetic peptide)
Average holdup BI: 0.25
Holdup BI standard deviation: 0.15
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): 1.58
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 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: KSR1
Protein B fragment: 268-277
Protein B site: 14-3-3_pThr273
Protein B modification: (TPO)273
Protein B sequence: FITPPTTPQL
Protein B construct: FITPP(pThr)TPQL (crude synthetic peptide)
Average holdup BI: -0.03
Holdup BI standard deviation: 0.24
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): 0.08
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: 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: KSR1
Protein B fragment: 326-335
Protein B site: 14-3-3_pSer331
Protein B modification: (SEP)331
Protein B sequence: MRFDLSHGSP
Protein B construct: MRFDL(pSer)HGSP (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.14
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): 0.07
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 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: KSR1
Protein B fragment: 883-892
Protein B site: 14-3-3_pSer888
Protein B modification: (SEP)888
Protein B sequence: LNRRLSHPGH
Protein B construct: LNRRL(pSer)HPGH (crude synthetic peptide)
Average holdup BI: -0.18
Holdup BI standard deviation: 2.11
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): 0.06
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: KSR1
Protein B fragment: 268-277
Protein B site: 14-3-3_pThr273
Protein B modification: (TPO)273
Protein B sequence: FITPPTTPQL
Protein B construct: FITPP(pThr)TPQL (crude synthetic peptide)
Average holdup BI: -0.02
Holdup BI standard deviation: 0.3
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): 0.04
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: 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: KSR1
Protein B fragment: 306-315
Protein B site: 14-3-3_pSer311
Protein B modification: (SEP)311
Protein B sequence: LTRSKSHESQ
Protein B construct: LTRSK(pSer)HESQ (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.57
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): 0.01
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.