Details of experiments between the fragments P61981~1-247 and Q9UQC2~205-214
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: GAB2
Protein B fragment: 205-214
Protein B site: 14-3-3_pSer210
Protein B modification: (SEP)210
Protein B sequence: NARSASFSQG
Protein B construct: NARSA(pSer)FSQG (crude synthetic peptide)
Average holdup BI: 0.37
Holdup BI standard deviation: 0.05
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): 4.91
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 2
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: GAB2
Protein B fragment: 205-214
Protein B site: 14-3-3_pSer210
Protein B modification: (SEP)210
Protein B sequence: NARSASFSQG
Protein B construct: NARSA(pSer)FSQG (crude synthetic peptide)
Average holdup BI: 0.5
Holdup BI standard deviation: 0.12
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): 4.08
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: 3.89
Experiment series 3
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: GAB2
Protein B fragment: 205-214
Protein B site: 14-3-3_pSer210
Protein B modification: (SEP)210
Protein B sequence: NARSASFSQG
Protein B construct: NARSA(pSer)FSQG (crude synthetic peptide)
Average holdup BI: 0.28
Holdup BI standard deviation: 0.05
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): 4.07
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
Measured pKd: 3.55