Details of experiments between Q99759 and P63104
Note that the results of all experiments are listed, regardless of the modification states of the fragments.



Experiment series 1

Protein A protein: MAP3K3
Protein A fragment: 245-254
Protein A site: 14-3-3_pSer250
Protein A modification: (SEP)250
Protein A sequence: MSRAQSFPDN
Protein A construct: MSRAQ(pSer)FPDN (crude synthetic peptide)

Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta

Average holdup BI: 0.7
Holdup BI standard deviation: 0.01
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): 12.07
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: 4.25


Experiment series 2

Protein A protein: MAP3K3
Protein A fragment: 352-361
Protein A site: 14-3-3_pSer357
Protein A modification: (SEP)357
Protein A sequence: PTKSPSAPIN
Protein A construct: PTKSP(pSer)APIN (crude synthetic peptide)

Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta

Average holdup BI: 0.74
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): 7.63
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: 4.33


Experiment series 3

Protein A protein: MAP3K3
Protein A fragment: 289-298
Protein A site: 14-3-3_pThr294
Protein A modification: (TPO)294
Protein A sequence: SDGRRTFPRI
Protein A construct: SDGRR(pThr)FPRI (crude synthetic peptide)

Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta

Average holdup BI: 0.8
Holdup BI standard deviation: 0.03
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): 6.69
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: 4.47


Experiment series 4

Protein A protein: MAP3K3
Protein A fragment: 523-532
Protein A site: 14-3-3_pThr528
Protein A modification: (TPO)528
Protein A sequence: GMRSVTGTPY
Protein A construct: GMRSV(pThr)GTPY (crude synthetic peptide)

Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta

Average holdup BI: 0.58
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): 6.2
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: 4.02


Experiment series 5

Protein A protein: MAP3K3
Protein A fragment: 142-151
Protein A site: 14-3-3_pSer147
Protein A modification: (SEP)147
Protein A sequence: IKASQSAGDI
Protein A construct: IKASQ(pSer)AGDI (crude synthetic peptide)

Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta

Average holdup BI: 0.04
Holdup BI standard deviation: 0.05
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): 0.78
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 6

Protein A protein: MAP3K3
Protein A fragment: 335-344
Protein A site: 14-3-3_pSer340
Protein A modification: (SEP)340
Protein A sequence: LRSADSENAL
Protein A construct: LRSAD(pSer)ENAL (crude synthetic peptide)

Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta

Average holdup BI: 0.03
Holdup BI standard deviation: 0.06
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): 0.41
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
The affinity was below the detection threshold of the assay.


Experiment series 7

Protein A protein: MAP3K3
Protein A fragment: 312-321
Protein A site: 14-3-3_pThr317
Protein A modification: (TPO)317
Protein A sequence: SRSLSTNGEN
Protein A construct: SRSLS(pThr)NGEN (crude synthetic peptide)

Protein B protein: YWHAZ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3zeta

Average holdup BI: -0.46
Holdup BI standard deviation: 3.94
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): 0.11
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.