Details of experiments between Q9UQC2 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: GAB2
Protein A fragment: 128-137
Protein A site: 14-3-3_pSer133
Protein A modification: (SEP)133
Protein A sequence: LRNVSSAGHG
Protein A construct: LRNVS(pSer)AGHG (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): <100
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: 4


Experiment series 2

Protein A protein: GAB2
Protein A fragment: 380-389
Protein A site: 14-3-3_pThr385
Protein A modification: (TPO)385
Protein A sequence: RSVAATIPRR
Protein A construct: RSVAA(pThr)IPRR (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: 0.72
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): 7.89
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: 4.28


Experiment series 3

Protein A protein: GAB2
Protein A fragment: 386-395
Protein A site: 14-3-3_pThr391
Protein A modification: (TPO)391
Protein A sequence: IPRRNTLPAM
Protein A construct: IPRRN(pThr)LPAM (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: 0.96
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): 6.9
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: 5.22


Experiment series 4

Protein A protein: GAB2
Protein A fragment: 207-216
Protein A site: 14-3-3_pSer212
Protein A modification: (SEP)212
Protein A sequence: RSASFSQGTR
Protein A construct: RSASF(pSer)QGTR (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: 0.39
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): 4.34
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: 3.68


Experiment series 5

Protein A protein: GAB2
Protein A fragment: 205-214
Protein A site: 14-3-3_pSer210
Protein A modification: (SEP)210
Protein A sequence: NARSASFSQG
Protein A construct: NARSA(pSer)FSQG (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: 0.37
Holdup BI standard deviation: 0.14
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): 2.96
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: 3.65


Experiment series 6

Protein A protein: GAB2
Protein A fragment: 259-268
Protein A site: 14-3-3_pSer264
Protein A modification: (SEP)264
Protein A sequence: TEFRDSTYDL
Protein A construct: TEFRD(pSer)TYDL (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: -0.07
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): 1.18
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: GAB2
Protein A fragment: 467-476
Protein A site: 14-3-3_pSer472
Protein A modification: (SEP)472
Protein A sequence: RAGDNSQSVY
Protein A construct: RAGDN(pSer)QSVY (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): 0.43
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: GAB2
Protein A fragment: 317-326
Protein A site: 14-3-3_pSer322
Protein A modification: (SEP)322
Protein A sequence: PATPLSAYQI
Protein A construct: PATPL(pSer)AYQI (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: 0.13
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 9

Protein A protein: GAB2
Protein A fragment: 207-216
Protein A site: 14-3-3_pSer212
Protein A modification: (SEP)212
Protein A sequence: RSASFSQGTR
Protein A construct: biotin-ttds-RSASF(pSer)QGTR (HPLC-purified 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): 195
Standard deviation of dissociation constant (10-6M): 7
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit

Date of measurement: 2022.01.19
Number of measurements: 3
Measured pKd: 3.71


Experiment series 10

Protein A protein: GAB2
Protein A fragment: 207-216
Protein A site: 14-3-3_pSer212
Protein A modification: (SEP)212
Protein A sequence: RSASFSQGTR
Protein A construct: biotin-ttds-RSASF(pSer)QGTR (HPLC-purified 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): 166
Standard deviation of dissociation constant (10-6M): 53
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit

Date of measurement: 2022.02.10
Number of measurements: 3
Measured pKd: 3.78