Details of experiments between P63104 and Q7Z401
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: DENND4A
Protein B fragment: 1585-1594
Protein B site: 14-3-3_pThr1590
Protein B modification: (TPO)1590
Protein B sequence: ARSISTSGPL
Protein B construct: ARSIS(pThr)SGPL (crude synthetic peptide)
Average holdup BI: 0.46
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.69
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: 3.81
Experiment series 2
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: DENND4A
Protein B fragment: 1503-1512
Protein B site: 14-3-3_pSer1508
Protein B modification: (SEP)1508
Protein B sequence: RYFLKSSPST
Protein B construct: RYFLK(pSer)SPST (crude synthetic peptide)
Average holdup BI: 0.7
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.22
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: 4.26
Experiment series 3
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: DENND4A
Protein B fragment: 1586-1595
Protein B site: 14-3-3_pSer1591
Protein B modification: (SEP)1591
Protein B sequence: RSISTSGPLD
Protein B construct: RSIST(pSer)GPLD (crude synthetic peptide)
Average holdup BI: 0.19
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.83
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 4
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: DENND4A
Protein B fragment: 1276-1285
Protein B site: 14-3-3_pSer1281
Protein B modification: (SEP)1281
Protein B sequence: RDRLWSSPAF
Protein B construct: RDRLW(pSer)SPAF (crude synthetic peptide)
Average holdup BI: 0.92
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.79
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: 4.94
Experiment series 5
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: DENND4A
Protein B fragment: 1030-1039
Protein B site: 14-3-3_pSer1035
Protein B modification: (SEP)1035
Protein B sequence: RNRNLSGGVL
Protein B construct: RNRNL(pSer)GGVL (crude synthetic peptide)
Average holdup BI: 0.25
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.16
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: 3.39
Experiment series 6
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: DENND4A
Protein B fragment: 1146-1155
Protein B site: 14-3-3_pSer1151
Protein B modification: (SEP)1151
Protein B sequence: IQRMNSSFSV
Protein B construct: IQRMN(pSer)SFSV (crude synthetic peptide)
Average holdup BI: 0.13
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.81
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 7
Protein A protein: YWHAZ
Protein A fragment: 1-245
Protein A site: 14-3-3
Protein A construct: avi-his6-MBP-TEVsite-14-3-3zeta
Protein B protein: DENND4A
Protein B fragment: 1220-1229
Protein B site: 14-3-3_pSer1225
Protein B modification: (SEP)1225
Protein B sequence: TSRTPSIDLQ
Protein B construct: TSRTP(pSer)IDLQ (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 132
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.23
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.