Details of experiments between the fragments Q7Z460~277-286 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: CLASP1
Protein A fragment: 277-286
Protein A site: 14-3-3_pThr282
Protein A modification: (TPO)282
Protein A sequence: RLGSSTLGSK
Protein A construct: RLGSS(pThr)LGSK (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: 0.35
Holdup BI standard deviation: 0.06
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): 4.85
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: 3.58
Experiment series 2
Protein A protein: CLASP1
Protein A fragment: 277-286
Protein A site: 14-3-3_pThr282
Protein A modification: (TPO)282
Protein A sequence: RLGSSTLGSK
Protein A construct: RLGSS(pThr)LGSK (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: 0.37
Holdup BI standard deviation: 0.11
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): 3.15
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: 3.72
Experiment series 3
Protein A protein: CLASP1
Protein A fragment: 277-286
Protein A site: 14-3-3_pThr282
Protein A modification: (TPO)282
Protein A sequence: RLGSSTLGSK
Protein A construct: RLGSS(pThr)LGSK (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: 0.15
Holdup BI standard deviation: 0.3
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): 0.37
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
The affinity was below the detection threshold of the assay.