Details of experiments between the fragments P62258~1-255 and O43166~1399-1408
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: SIPA1L1
Protein B fragment: 1399-1408
Protein B site: 14-3-3_pSer1404
Protein B modification: (SEP)1404
Protein B sequence: KSQAGSTPLT
Protein B construct: KSQAG(pSer)TPLT (crude synthetic peptide)
Average holdup BI: 0.15
Holdup BI standard deviation: 0.04
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): 3.58
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: 3.09
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: SIPA1L1
Protein B fragment: 1399-1408
Protein B site: 14-3-3_pSer1404
Protein B modification: (SEP)1404
Protein B sequence: KSQAGSTPLT
Protein B construct: KSQAG(pSer)TPLT (crude synthetic peptide)
Average holdup BI: 0.09
Holdup BI standard deviation: 0.03
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): 2.88
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.