Details of experiments between P27348 and Q32P44
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: EML3
Protein B fragment: 171-180
Protein B site: 14-3-3_pSer176
Protein B modification: (SEP)176
Protein B sequence: SRKAISSANL
Protein B construct: SRKAI(pSer)SANL (crude synthetic peptide)
Average holdup BI: 0.55
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 8.24
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: 3.97
Experiment series 2
Protein A protein: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: EML3
Protein B fragment: 192-201
Protein B site: 14-3-3_pSer197
Protein B modification: (SEP)197
Protein B sequence: GKDPLSSPGG
Protein B construct: GKDPL(pSer)SPGG (crude synthetic peptide)
Average holdup BI: 0.2
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.21
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: 3.27
Experiment series 3
Protein A protein: YWHAQ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3tau
Protein B protein: EML3
Protein B fragment: 202-211
Protein B site: 14-3-3_pSer207
Protein B modification: (SEP)207
Protein B sequence: PGSRRSNYNL
Protein B construct: PGSRR(pSer)NYNL (crude synthetic peptide)
Average holdup BI: 0.04
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.87
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
The affinity was below the detection threshold of the assay.