Details of experiments between P63104 and O60282
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: KIF5C
Protein B fragment: 929-938
Protein B site: 14-3-3_pSer934
Protein B modification: (SEP)934
Protein B sequence: GHYPASSPTA
Protein B construct: GHYPA(pSer)SPTA (crude synthetic peptide)
Average holdup BI: 0.64
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): 6.25
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
Measured pKd: 4.12
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: KIF5C
Protein B fragment: 312-321
Protein B site: 14-3-3_pThr317
Protein B modification: (TPO)317
Protein B sequence: AETKSTLMFG
Protein B construct: AETKS(pThr)LMFG (crude synthetic peptide)
Average holdup BI: 0.07
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): 1.76
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 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: KIF5C
Protein B fragment: 811-820
Protein B site: 14-3-3_pSer816
Protein B modification: (SEP)816
Protein B sequence: TRVKKSVELD
Protein B construct: TRVKK(pSer)VELD (crude synthetic peptide)
Average holdup BI: -0.19
Holdup BI standard deviation: 0.28
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): 0.93
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 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: KIF5C
Protein B fragment: 655-664
Protein B site: 14-3-3_pSer660
Protein B modification: (SEP)660
Protein B sequence: RQLEESQDSL
Protein B construct: RQLEE(pSer)QDSL (crude synthetic peptide)
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): 0.65
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: KIF5C
Protein B fragment: 591-600
Protein B site: 14-3-3_pSer596
Protein B modification: (SEP)596
Protein B sequence: ISKMKSEVKS
Protein B construct: ISKMK(pSer)EVKS (crude synthetic peptide)
Average holdup BI: 0.14
Holdup BI standard deviation: 0.24
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): 0.54
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: 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: KIF5C
Protein B fragment: 605-614
Protein B site: 14-3-3_pSer610
Protein B modification: (SEP)610
Protein B sequence: SKQLESAQMD
Protein B construct: SKQLE(pSer)AQMD (crude synthetic peptide)
Average holdup BI: 0.03
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): 0.37
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
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: KIF5C
Protein B fragment: 610-619
Protein B site: 14-3-3_pSer615
Protein B modification: (SEP)615
Protein B sequence: SAQMDSNRKM
Protein B construct: SAQMD(pSer)NRKM (crude synthetic peptide)
Average holdup BI: 0.18
Holdup BI standard deviation: 0.59
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): 0.23
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: KIF5C
Protein B fragment: 830-839
Protein B site: 14-3-3_pSer835
Protein B modification: (SEP)835
Protein B sequence: QKQKISFLEN
Protein B construct: QKQKI(pSer)FLEN (crude synthetic peptide)
Average holdup BI: 0.01
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): 0.13
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.