Details of experiments between the fragments P31947~1-248 and Q6R327~1279-1288
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: RICTOR
Protein B fragment: 1279-1288
Protein B site: 14-3-3_pSer1284
Protein B modification: (SEP)1284
Protein B sequence: LSKSNSVSLV
Protein B construct: LSKSN(pSer)VSLV (crude synthetic peptide)
Average holdup BI: 0.22
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 113
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.8
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: 3.39
Experiment series 2
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: RICTOR
Protein B fragment: 1279-1288
Protein B site: 14-3-3_pSer1284
Protein B modification: (SEP)1284
Protein B sequence: LSKSNSVSLV
Protein B construct: LSKSN(pSer)VSLV (crude synthetic peptide)
Average holdup BI: 0.25
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 142
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.95
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: 3.38
Experiment series 3
Protein A protein: SFN
Protein A fragment: 1-248
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3sigma
Protein B protein: RICTOR
Protein B fragment: 1279-1288
Protein B site: 14-3-3_pSer1284
Protein B modification: (SEP)1284
Protein B sequence: LSKSNSVSLV
Protein B construct: LSKSN(pSer)VSLV (crude synthetic peptide)
Average holdup BI: 0.27
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 111
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.89
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: 3.51