Details of experiments between the fragments Q96PY6~409-418 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: NEK1
Protein A fragment: 409-418
Protein A site: 14-3-3_pSer414
Protein A modification: (SEP)414
Protein A sequence: WRNVLSAGGS
Protein A construct: WRNVL(pSer)AGGS (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: 0.29
Holdup BI standard deviation: 0.06
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): 3.57
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: 3.56
Experiment series 2
Protein A protein: NEK1
Protein A fragment: 409-418
Protein A site: 14-3-3_pSer414
Protein A modification: (SEP)414
Protein A sequence: WRNVLSAGGS
Protein A construct: WRNVL(pSer)AGGS (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: 0.23
Holdup BI standard deviation: 0.08
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): 3.04
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: 3.33
Experiment series 3
Protein A protein: NEK1
Protein A fragment: 409-418
Protein A site: 14-3-3_pSer414
Protein A modification: (SEP)414
Protein A sequence: WRNVLSAGGS
Protein A construct: WRNVL(pSer)AGGS (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: 0.17
Holdup BI standard deviation: 0.06
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): 2.88
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.