Details of experiments between the fragments Q6R327~1214-1223 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: RICTOR
Protein A fragment: 1214-1223
Protein A site: 14-3-3_pSer1219
Protein A modification: (SEP)1219
Protein A sequence: KIRSQSFNTD
Protein A construct: KIRSQ(pSer)FNTD (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: 0.55
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): 8.39
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: 4.08
Experiment series 2
Protein A protein: RICTOR
Protein A fragment: 1214-1223
Protein A site: 14-3-3_pSer1219
Protein A modification: (SEP)1219
Protein A sequence: KIRSQSFNTD
Protein A construct: KIRSQ(pSer)FNTD (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: 0.52
Holdup BI standard deviation: 0.02
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): 7.09
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: 3.91
Experiment series 3
Protein A protein: RICTOR
Protein A fragment: 1214-1223
Protein A site: 14-3-3_pSer1219
Protein A modification: (SEP)1219
Protein A sequence: KIRSQSFNTD
Protein A construct: biotin-ttds-KIRSQ(pSer)FNTD (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): 121
Standard deviation of dissociation constant (10-6M): 38
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: 3.92