Details of experiments between the fragments Q12778~271-280 and P61981~1-247
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: FOXO1
Protein A fragment: 271-280
Protein A site: 14-3-3_pSer276
Protein A modification: (SEP)276
Protein A sequence: AKKKASLQSG
Protein A construct: AKKKA(pSer)LQSG (crude synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Average holdup BI: 0.56
Holdup BI standard deviation: 0.16
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.63
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: 3.99
Experiment series 2
Protein A protein: FOXO1
Protein A fragment: 271-280
Protein A site: 14-3-3_pSer276
Protein A modification: (SEP)276
Protein A sequence: AKKKASLQSG
Protein A construct: AKKKA(pSer)LQSG (crude synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Average holdup BI: 0.75
Holdup BI standard deviation: 0.23
Immobilized partner concentration in holdup experiment (10-6M): 115
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: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
Measured pKd: 4.44
Experiment series 3
Protein A protein: FOXO1
Protein A fragment: 271-280
Protein A site: 14-3-3_pSer276
Protein A modification: (SEP)276
Protein A sequence: AKKKASLQSG
Protein A construct: AKKKA(pSer)LQSG (crude synthetic peptide)
Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma
Average holdup BI: -0.14
Holdup BI standard deviation: 1.02
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): 0.07
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.