Details of experiments between O75044 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: SRGAP2
Protein A fragment: 1065-1074
Protein A site: 14-3-3_pThr1070
Protein A modification: (TPO)1070
Protein A sequence: TDKSCTV---
Protein A construct: PQSTDKSC(pThr)V (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
Measured dissociation constant (10-6M): <100
Immobilized partner concentration in holdup experiment (10-6M): 116
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: SRGAP2
Protein A fragment: 422-431
Protein A site: 14-3-3_pSer427
Protein A modification: (SEP)427
Protein A sequence: VKSTVSETFM
Protein A construct: VKSTV(pSer)ETFM (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: 0.19
Holdup BI standard deviation: 0.08
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): 2.69
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: 3.21
Experiment series 3
Protein A protein: SRGAP2
Protein A fragment: 849-858
Protein A site: 14-3-3_pSer854
Protein A modification: (SEP)854
Protein A sequence: TFRSDSHGLS
Protein A construct: TFRSD(pSer)HGLS (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: 0.06
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): 1.51
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: SRGAP2
Protein A fragment: 1009-1018
Protein A site: 14-3-3_pThr1014
Protein A modification: (TPO)1014
Protein A sequence: QRSASTAGDI
Protein A construct: QRSAS(pThr)AGDI (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: 0.11
Holdup BI standard deviation: 0.24
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): 0.43
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 5
Protein A protein: SRGAP2
Protein A fragment: 849-858
Protein A site: 14-3-3_pSer854
Protein A modification: (SEP)854
Protein A sequence: TFRSDSHGLS
Protein A construct: TFRSD(pSer)HGLS (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: -0.02
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): 0.28
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.