Details of experiments between Q04917 and P56524
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: HDAC4
Protein B fragment: 260-269
Protein B site: 14-3-3_pSer265
Protein B modification: (SEP)265
Protein B sequence: VAERRSSPLL
Protein B construct: VAERR(pSer)SPLL (crude synthetic peptide)
Average holdup BI: 0.9
Holdup BI standard deviation: 0.04
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): 7.49
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: 4.83
Experiment series 2
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: HDAC4
Protein B fragment: 627-636
Protein B site: 14-3-3_pSer632
Protein B modification: (SEP)632
Protein B sequence: LSRAQSSPAS
Protein B construct: LSRAQ(pSer)SPAS (crude synthetic peptide)
Average holdup BI: 0.94
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): 4.45
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: 5.11
Experiment series 3
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: HDAC4
Protein B fragment: 345-354
Protein B site: 14-3-3_pSer350
Protein B modification: (SEP)350
Protein B sequence: LYTSPSLPNI
Protein B construct: LYTSP(pSer)LPNI (crude synthetic peptide)
Average holdup BI: 0.88
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): 3.41
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: 4.73
Experiment series 4
Protein A protein: YWHAH
Protein A fragment: 1-246
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3eta
Protein B protein: HDAC4
Protein B fragment: 579-588
Protein B site: 14-3-3_pSer584
Protein B modification: (SEP)584
Protein B sequence: GQRQPSEQEL
Protein B construct: GQRQP(pSer)EQEL (crude synthetic peptide)
Average holdup BI: -0.03
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): 1.01
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.