Details of experiments between the fragments Q9Y2J2~396-405 and P31946~1-246
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: EPB41L3
Protein A fragment: 396-405
Protein A site: 14-3-3_pThr401
Protein A modification: (TPO)401
Protein A sequence: PKKFLTLGSK
Protein A construct: PKKFL(pThr)LGSK (crude synthetic peptide)
Protein B protein: YWHAB
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3beta
Average holdup BI: 0.75
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.87
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: 4.47
Experiment series 2
Protein A protein: EPB41L3
Protein A fragment: 396-405
Protein A site: 14-3-3_pThr401
Protein A modification: (TPO)401
Protein A sequence: PKKFLTLGSK
Protein A construct: PKKFL(pThr)LGSK (crude synthetic peptide)
Protein B protein: YWHAB
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3beta
Average holdup BI: 0.77
Holdup BI standard deviation: 0.06
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): 5.33
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: 4.41
Experiment series 3
Protein A protein: EPB41L3
Protein A fragment: 396-405
Protein A site: 14-3-3_pThr401
Protein A modification: (TPO)401
Protein A sequence: PKKFLTLGSK
Protein A construct: biotin-ttds-PKKFL(pThr)LGSK (HPLC-purified synthetic peptide)
Protein B protein: YWHAB
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3beta
Measured dissociation constant (10-6M): 19
Standard deviation of dissociation constant (10-6M): 0.5
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022.02.08
Number of measurements: 3
Measured pKd: 4.73