Details of experiments between Q9UJ41 and P62258
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: RABGEF1
Protein A fragment: 101-110
Protein A site: 14-3-3_pSer106
Protein A modification: (SEP)106
Protein A sequence: VKKFFSASSR
Protein A construct: VKKFF(pSer)ASSR (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.47
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.78
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: 3.88
Experiment series 2
Protein A protein: RABGEF1
Protein A fragment: 101-110
Protein A site: 14-3-3_pSer106
Protein A modification: (SEP)106
Protein A sequence: VKKFFSASSR
Protein A construct: VKKFF(pSer)ASSR (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.48
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.52
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: 3.81
Experiment series 3
Protein A protein: RABGEF1
Protein A fragment: 372-381
Protein A site: 14-3-3_pSer377
Protein A modification: (SEP)377
Protein A sequence: QSLNLSQEDF
Protein A construct: QSLNL(pSer)QEDF (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.17
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.43
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: RABGEF1
Protein A fragment: 127-136
Protein A site: 14-3-3_pSer132
Protein A modification: (SEP)132
Protein A sequence: INRQTSIETD
Protein A construct: INRQT(pSer)IETD (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.05
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.31
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 5
Protein A protein: RABGEF1
Protein A fragment: 372-381
Protein A site: 14-3-3_pSer377
Protein A modification: (SEP)377
Protein A sequence: QSLNLSQEDF
Protein A construct: QSLNL(pSer)QEDF (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.08
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.98
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: RABGEF1
Protein A fragment: 127-136
Protein A site: 14-3-3_pSer132
Protein A modification: (SEP)132
Protein A sequence: INRQTSIETD
Protein A construct: INRQT(pSer)IETD (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.04
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.73
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: RABGEF1
Protein A fragment: 384-393
Protein A site: 14-3-3_pThr389
Protein A modification: (TPO)389
Protein A sequence: YMSGQTSPRK
Protein A construct: YMSGQ(pThr)SPRK (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.24
Holdup BI standard deviation: 0.22
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.69
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: RABGEF1
Protein A fragment: 108-117
Protein A site: 14-3-3_pSer113
Protein A modification: (SEP)113
Protein A sequence: SSRVGSKKEI
Protein A construct: SSRVG(pSer)KKEI (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.04
Holdup BI standard deviation: 0.13
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.23
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 9
Protein A protein: RABGEF1
Protein A fragment: 384-393
Protein A site: 14-3-3_pThr389
Protein A modification: (TPO)389
Protein A sequence: YMSGQTSPRK
Protein A construct: YMSGQ(pThr)SPRK (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.01
Holdup BI standard deviation: 0.63
Immobilized partner concentration in holdup experiment (10-6M): 143
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: 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.