Details of experiments between the fragments P61981~1-247 and Q9P0K7~291-300
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: RAI14
Protein B fragment: 291-300
Protein B site: 14-3-3_pSer296
Protein B modification: (SEP)296
Protein B sequence: PRSITSTPLS
Protein B construct: PRSIT(pSer)TPLS (crude synthetic peptide)
Average holdup BI: 0.55
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): 6.59
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: 3.96
Experiment series 2
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A modification: 100_uM_FCA
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: RAI14
Protein B fragment: 291-300
Protein B site: 14-3-3_pSer296
Protein B modification: (SEP)296
Protein B sequence: PRSITSTPLS
Protein B construct: PRSIT(pSer)TPLS (crude synthetic peptide)
Average holdup BI: 0.44
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 115
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 6.12
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: 3.85
Experiment series 3
Protein A protein: YWHAG
Protein A fragment: 1-247
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3gamma
Protein B protein: RAI14
Protein B fragment: 291-300
Protein B site: 14-3-3_pSer296
Protein B modification: (SEP)296
Protein B sequence: PRSITSTPLS
Protein B construct: PRSIT(pSer)TPLS (crude synthetic peptide)
Average holdup BI: 0.52
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 129
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: 2021.10.13.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 5))
Number of measurements: 5
Measured pKd: 3.92