Details of experiments between the fragments P62258~1-255 and Q5T5U3~454-463
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Protein B protein: ARHGAP21
Protein B fragment: 454-463
Protein B site: 14-3-3_pSer459
Protein B modification: (SEP)459
Protein B sequence: RQRSVSQERL
Protein B construct: RQRSV(pSer)QERL (crude synthetic peptide)
Average holdup BI: 0.19
Holdup BI standard deviation: 0.12
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): 1.65
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
Measured pKd: 3.2
Experiment series 2
Protein A protein: YWHAE
Protein A fragment: 1-255
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Protein B protein: ARHGAP21
Protein B fragment: 454-463
Protein B site: 14-3-3_pSer459
Protein B modification: (SEP)459
Protein B sequence: RQRSVSQERL
Protein B construct: RQRSV(pSer)QERL (crude synthetic peptide)
Average holdup BI: -0.19
Holdup BI standard deviation: 0.17
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): 1.29
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.