Details of experiments between P15056 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: BRAF
Protein A fragment: 360-369
Protein A site: 14-3-3_pSer365
Protein A modification: (SEP)365
Protein A sequence: RDRSSSAPNV
Protein A construct: RDRSS(pSer)APNV (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

Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 132
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: 1
Measured pKd: 5


Experiment series 2

Protein A protein: BRAF
Protein A fragment: 724-733
Protein A site: 14-3-3_pSer729
Protein A modification: (SEP)729
Protein A sequence: IHRSASEPSL
Protein A construct: IHRSA(pSer)EPSL (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: 0.95
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): 6.06
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: 5.17


Experiment series 3

Protein A protein: BRAF
Protein A fragment: 465-474
Protein A site: 14-3-3_pThr470
Protein A modification: (TPO)470
Protein A sequence: SGSFGTVYKG
Protein A construct: SGSFG(pThr)VYKG (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: 0.34
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): 6.01
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
Measured pKd: 3.59


Experiment series 4

Protein A protein: BRAF
Protein A fragment: 334-343
Protein A site: 14-3-3_pSer339
Protein A modification: (SEP)339
Protein A sequence: PSPSKSIPIP
Protein A construct: PSPSK(pSer)IPIP (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: 0.33
Holdup BI standard deviation: 0.07
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): 4.48
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 5

Protein A protein: BRAF
Protein A fragment: 462-471
Protein A site: 14-3-3_pSer467
Protein A modification: (SEP)467
Protein A sequence: RIGSGSFGTV
Protein A construct: RIGSG(pSer)FGTV (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: 0.14
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): 3.25
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: BRAF
Protein A fragment: 609-618
Protein A site: 14-3-3_pSer614
Protein A modification: (SEP)614
Protein A sequence: QFEQLSGSIL
Protein A construct: QFEQL(pSer)GSIL (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: -0.25
Holdup BI standard deviation: 0.31
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): 1.1
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: BRAF
Protein A fragment: 389-398
Protein A site: 14-3-3_pSer394
Protein A modification: (SEP)394
Protein A sequence: RGDGGSTTGL
Protein A construct: RGDGG(pSer)TTGL (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: 0.07
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): 0.75
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 8

Protein A protein: BRAF
Protein A fragment: 441-450
Protein A site: 14-3-3_pSer446
Protein A modification: (SEP)446
Protein A sequence: LGRRDSSDDW
Protein A construct: LGRRD(pSer)SDDW (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: -0.05
Holdup BI standard deviation: 0.07
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): 0.7
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 9

Protein A protein: BRAF
Protein A fragment: 594-603
Protein A site: 14-3-3_pThr599
Protein A modification: (TPO)599
Protein A sequence: DFGLATVKSR
Protein A construct: DFGLA(pThr)VKSR (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: 0.08
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): 0.38
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 10

Protein A protein: BRAF
Protein A fragment: 574-583
Protein A site: 14-3-3_pSer579
Protein A modification: (SEP)579
Protein A sequence: HRDLKSNNIF
Protein A construct: HRDLK(pSer)NNIF (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: 0.14
Holdup BI standard deviation: 0.39
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): 0.31
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 11

Protein A protein: BRAF
Protein A fragment: 597-606
Protein A site: 14-3-3_pSer602
Protein A modification: (SEP)602
Protein A sequence: LATVKSRWSG
Protein A construct: LATVK(pSer)RWSG (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
Holdup BI standard deviation: 0.07
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): 0.03
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.