Details of experiments between the fragments P61981~1-247 and Q9Y6M7~163-172
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: SLC4A7
Protein B fragment: 163-172
Protein B site: 14-3-3_pSer168
Protein B modification: (SEP)168
Protein B sequence: LASPQSAPGN
Protein B construct: LASPQ(pSer)APGN (crude synthetic peptide)
Average holdup BI: 0.73
Holdup BI standard deviation: 0.03
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): 8.23
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: 4.31
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: SLC4A7
Protein B fragment: 163-172
Protein B site: 14-3-3_pSer168
Protein B modification: (SEP)168
Protein B sequence: LASPQSAPGN
Protein B construct: LASPQ(pSer)APGN (crude synthetic peptide)
Average holdup BI: 0.7
Holdup BI standard deviation: 0.06
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): 6.15
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: 4.31
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: SLC4A7
Protein B fragment: 163-172
Protein B site: 14-3-3_pSer168
Protein B modification: (SEP)168
Protein B sequence: LASPQSAPGN
Protein B construct: LASPQ(pSer)APGN (crude synthetic peptide)
Average holdup BI: 0.81
Holdup BI standard deviation: 0.08
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): 4.37
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: 4.5