Details of experiments between Q86X27 and P61981
Note that the results of all experiments are listed, regardless of the modification states of the fragments.


Experiment series 1

Protein A protein: RALGPS2
Protein A fragment: 417-426
Protein A site: 14-3-3_pSer422
Protein A modification: (SEP)422
Protein A sequence: NRLYHSLGPV
Protein A construct: NRLYH(pSer)LGPV (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Measured dissociation constant (10-6M): <10
Immobilized partner concentration in holdup experiment (10-6M): 115
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 1
Measured pKd: 5

Experiment series 2

Protein A protein: RALGPS2
Protein A fragment: 356-365
Protein A site: 14-3-3_pThr361
Protein A modification: (TPO)361
Protein A sequence: EFKSATFPNA
Protein A construct: EFKSA(pThr)FPNA (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: 0.94
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 10.2
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
Measured pKd: 5.05

Experiment series 3

Protein A protein: RALGPS2
Protein A fragment: 356-365
Protein A site: 14-3-3_pThr361
Protein A modification: (TPO)361
Protein A sequence: EFKSATFPNA
Protein A construct: EFKSA(pThr)FPNA (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: 0.91
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 115
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 9.23
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
Measured pKd: 4.98

Experiment series 4

Protein A protein: RALGPS2
Protein A fragment: 356-365
Protein A site: 14-3-3_pThr361
Protein A modification: (TPO)361
Protein A sequence: EFKSATFPNA
Protein A construct: EFKSA(pThr)FPNA (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: 0.94
Holdup BI standard deviation: 0
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 9.2
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: 5.1

Experiment series 5

Protein A protein: RALGPS2
Protein A fragment: 417-426
Protein A site: 14-3-3_pSer422
Protein A modification: (SEP)422
Protein A sequence: NRLYHSLGPV
Protein A construct: NRLYH(pSer)LGPV (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: 0.95
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 7.04
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 6

Protein A protein: RALGPS2
Protein A fragment: 291-300
Protein A site: 14-3-3_pSer296
Protein A modification: (SEP)296
Protein A sequence: PRSAASREDL
Protein A construct: PRSAA(pSer)REDL (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: 0.03
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.57
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
The affinity was below the detection threshold of the assay.

Experiment series 7

Protein A protein: RALGPS2
Protein A fragment: 291-300
Protein A site: 14-3-3_pSer296
Protein A modification: (SEP)296
Protein A sequence: PRSAASREDL
Protein A construct: PRSAA(pSer)REDL (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: -0.03
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 129
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.39
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: RALGPS2
Protein A fragment: 291-300
Protein A site: 14-3-3_pSer296
Protein A modification: (SEP)296
Protein A sequence: PRSAASREDL
Protein A construct: PRSAA(pSer)REDL (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B modification: 100_uM_FCA
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: -0.06
Holdup BI standard deviation: 0.15
Immobilized partner concentration in holdup experiment (10-6M): 115
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.34
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
The affinity was below the detection threshold of the assay.

Experiment series 9

Protein A protein: RALGPS2
Protein A fragment: 417-426
Protein A site: 14-3-3_pSer422
Protein A modification: (SEP)422
Protein A sequence: NRLYHSLGPV
Protein A construct: NRLYH(pSer)LGPV (crude synthetic peptide)

Protein B protein: YWHAG
Protein B fragment: 1-247
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3gamma

Average holdup BI: 0.35
Holdup BI standard deviation: 1.07
Immobilized partner concentration in holdup experiment (10-6M): 134
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.28
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.02.17.
Experimental details: -log10(P Two-sided unpaired T-test (4 vs 4))
Number of measurements: 4
The affinity was below the detection threshold of the assay.