Details of experiments between P31946 and Q9H6H4
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: REEP4
Protein B fragment: 219-228
Protein B site: 14-3-3_pSer224
Protein B modification: (SEP)224
Protein B sequence: LIRSQSLRVV
Protein B construct: LIRSQ(pSer)LRVV (crude synthetic peptide)
Average holdup BI: 0.87
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 9.22
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: 4.8
Experiment series 2
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: REEP4
Protein B fragment: 219-228
Protein B site: 14-3-3_pSer224
Protein B modification: (SEP)224
Protein B sequence: LIRSQSLRVV
Protein B construct: LIRSQ(pSer)LRVV (crude synthetic peptide)
Average holdup BI: 0.89
Holdup BI standard deviation: 0.01
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): 5.63
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: 4.79
Experiment series 3
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: REEP4
Protein B fragment: 147-156
Protein B site: 14-3-3_pSer152
Protein B modification: (SEP)152
Protein B sequence: RLRSFSMQDL
Protein B construct: RLRSF(pSer)MQDL (crude synthetic peptide)
Average holdup BI: 0.87
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.45
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: 4.81
Experiment series 4
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: REEP4
Protein B fragment: 248-257
Protein B site: 14-3-3_pSer253
Protein B modification: (SEP)253
Protein B sequence: KKTVPSDVDS
Protein B construct: KKTVP(pSer)DVDS (crude synthetic peptide)
Average holdup BI: -0.12
Holdup BI standard deviation: 0.19
Immobilized partner concentration in holdup experiment (10-6M): 102
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.73
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.
Experiment series 5
Protein A protein: YWHAB
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3beta
Protein B protein: REEP4
Protein B fragment: 248-257
Protein B site: 14-3-3_pSer253
Protein B modification: (SEP)253
Protein B sequence: KKTVPSDVDS
Protein B construct: KKTVP(pSer)DVDS (crude synthetic peptide)
Average holdup BI: 0
Holdup BI standard deviation: 0.09
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): 0.01
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
The affinity was below the detection threshold of the assay.