Details of experiments between P63104 and Q7Z460
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: CLASP1
Protein B fragment: 1079-1088
Protein B site: 14-3-3_pThr1084
Protein B modification: (TPO)1084
Protein B sequence: RTSPLTSPTN
Protein B construct: RTSPL(pThr)SPTN (crude synthetic peptide)
Average holdup BI: 0.66
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): 10.5
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: 4.17
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: CLASP1
Protein B fragment: 277-286
Protein B site: 14-3-3_pThr282
Protein B modification: (TPO)282
Protein B sequence: RLGSSTLGSK
Protein B construct: RLGSS(pThr)LGSK (crude synthetic peptide)
Average holdup BI: 0.45
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): 6.87
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: 3.79
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: CLASP1
Protein B fragment: 682-691
Protein B site: 14-3-3_pSer687
Protein B modification: (SEP)687
Protein B sequence: KVVSQSQPGS
Protein B construct: KVVSQ(pSer)QPGS (crude synthetic peptide)
Average holdup BI: 0.62
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): 6.73
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: 4.09
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: CLASP1
Protein B fragment: 589-598
Protein B site: 14-3-3_pSer594
Protein B modification: (SEP)594
Protein B sequence: VSTTGSLQRS
Protein B construct: VSTTG(pSer)LQRS (crude synthetic peptide)
Average holdup BI: 0.16
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): 4.41
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 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: CLASP1
Protein B fragment: 631-640
Protein B site: 14-3-3_pSer636
Protein B modification: (SEP)636
Protein B sequence: PGSYASLGRI
Protein B construct: PGSYA(pSer)LGRI (crude synthetic peptide)
Average holdup BI: 0.32
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): 4.37
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: 3.55
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: CLASP1
Protein B fragment: 593-602
Protein B site: 14-3-3_pSer598
Protein B modification: (SEP)598
Protein B sequence: GSLQRSRSDI
Protein B construct: GSLQR(pSer)RSDI (crude synthetic peptide)
Average holdup BI: 0.07
Holdup BI standard deviation: 0.09
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): 0.83
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 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: CLASP1
Protein B fragment: 790-799
Protein B site: 14-3-3_pSer795
Protein B modification: (SEP)795
Protein B sequence: AMRVLSTSTD
Protein B construct: AMRVL(pSer)TSTD (crude synthetic peptide)
Average holdup BI: 0.02
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): 0.61
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: 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: CLASP1
Protein B fragment: 792-801
Protein B site: 14-3-3_pSer797
Protein B modification: (SEP)797
Protein B sequence: RVLSTSTDLE
Protein B construct: RVLST(pSer)TDLE (crude synthetic peptide)
Average holdup BI: 0
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): 0.17
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: 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: CLASP1
Protein B fragment: 1191-1200
Protein B site: 14-3-3_pSer1196
Protein B modification: (SEP)1196
Protein B sequence: KFSFRSQEDL
Protein B construct: KFSFR(pSer)QEDL (crude synthetic peptide)
Average holdup BI: 0.03
Holdup BI standard deviation: 0.16
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): 0.15
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: 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: CLASP1
Protein B fragment: 628-637
Protein B site: 14-3-3_pSer633
Protein B modification: (SEP)633
Protein B sequence: ALPPGSYASL
Protein B construct: ALPPG(pSer)YASL (crude synthetic peptide)
Average holdup BI: 0.02
Holdup BI standard deviation: 0.09
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): 0.14
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: 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: CLASP1
Protein B fragment: 563-572
Protein B site: 14-3-3_pSer568
Protein B modification: (SEP)568
Protein B sequence: LNRPLSAKRS
Protein B construct: LNRPL(pSer)AKRS (crude synthetic peptide)
Average holdup BI: 0.02
Holdup BI standard deviation: 0.73
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): 0.02
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 12
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: CLASP1
Protein B fragment: 682-691
Protein B site: 14-3-3_pSer687
Protein B modification: (SEP)687
Protein B sequence: KVVSQSQPGS
Protein B construct: biotin-ttds-KVVSQ(pSer)QPGS (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 109
Standard deviation of dissociation constant (10-6M): 18
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: 3.96
Experiment series 13
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: CLASP1
Protein B fragment: 682-691
Protein B site: 14-3-3_pSer687
Protein B modification: (SEP)687
Protein B sequence: KVVSQSQPGS
Protein B construct: biotin-ttds-KVVSQ(pSer)QPGS (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 101
Standard deviation of dissociation constant (10-6M): 15
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: 3.99