Details of experiments between the fragments P31946~1-246 and Q9P2M7~126-135
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: CGN
Protein B fragment: 126-135
Protein B site: 14-3-3_pSer131
Protein B modification: (SEP)131
Protein B sequence: LLRSHSQASL
Protein B construct: LLRSH(pSer)QASL (crude synthetic peptide)
Average holdup BI: 0.33
Holdup BI standard deviation: 0.03
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): 5.71
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: 3.57
Experiment series 2
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: CGN
Protein B fragment: 126-135
Protein B site: 14-3-3_pSer131
Protein B modification: (SEP)131
Protein B sequence: LLRSHSQASL
Protein B construct: LLRSH(pSer)QASL (crude synthetic peptide)
Average holdup BI: 0.28
Holdup BI standard deviation: 0.05
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): 4.59
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: 3.59
Experiment series 3
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: CGN
Protein B fragment: 126-135
Protein B site: 14-3-3_pSer131
Protein B modification: (SEP)131
Protein B sequence: LLRSHSQASL
Protein B construct: biotin-ttds-LLRSH(pSer)QASL (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 90
Standard deviation of dissociation constant (10-6M): 20
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: 4.05