Details of experiments between the fragments Q9UJ41~101-110 and P31947~1-248
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: RABGEF1
Protein A fragment: 101-110
Protein A site: 14-3-3_pSer106
Protein A modification: (SEP)106
Protein A sequence: VKKFFSASSR
Protein A construct: VKKFF(pSer)ASSR (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Average holdup BI: 0.54
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): 7.07
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: 4.02
Experiment series 2
Protein A protein: RABGEF1
Protein A fragment: 101-110
Protein A site: 14-3-3_pSer106
Protein A modification: (SEP)106
Protein A sequence: VKKFFSASSR
Protein A construct: VKKFF(pSer)ASSR (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Average holdup BI: 0.58
Holdup BI standard deviation: 0.06
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): 6.19
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: 3.98
Experiment series 3
Protein A protein: RABGEF1
Protein A fragment: 101-110
Protein A site: 14-3-3_pSer106
Protein A modification: (SEP)106
Protein A sequence: VKKFFSASSR
Protein A construct: VKKFF(pSer)ASSR (crude synthetic peptide)
Protein B protein: SFN
Protein B fragment: 1-248
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3sigma
Average holdup BI: 0.56
Holdup BI standard deviation: 0.07
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): 4.96
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: 4.05