Details of experiments between O60333 and P62258
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: KIF1B
Protein A fragment: 1136-1145
Protein A site: 14-3-3_pSer1141
Protein A modification: (SEP)1141
Protein A sequence: MRRQFSAPNL
Protein A construct: MRRQF(pSer)APNL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.84
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.83
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
Measured pKd: 4.57
Experiment series 2
Protein A protein: KIF1B
Protein A fragment: 1048-1057
Protein A site: 14-3-3_pSer1053
Protein A modification: (SEP)1053
Protein A sequence: RWRSNSLNNG
Protein A construct: RWRSN(pSer)LNNG (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.57
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.48
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: 3.97
Experiment series 3
Protein A protein: KIF1B
Protein A fragment: 1048-1057
Protein A site: 14-3-3_pSer1053
Protein A modification: (SEP)1053
Protein A sequence: RWRSNSLNNG
Protein A construct: RWRSN(pSer)LNNG (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.61
Holdup BI standard deviation: 0.09
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.81
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: 4.13
Experiment series 4
Protein A protein: KIF1B
Protein A fragment: 1136-1145
Protein A site: 14-3-3_pSer1141
Protein A modification: (SEP)1141
Protein A sequence: MRRQFSAPNL
Protein A construct: MRRQF(pSer)APNL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.77
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.93
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: 4.46
Experiment series 5
Protein A protein: KIF1B
Protein A fragment: 1052-1061
Protein A site: 14-3-3_pSer1057
Protein A modification: (SEP)1057
Protein A sequence: TRSGLSLEEL
Protein A construct: TRSGL(pSer)LEEL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.09
Holdup BI standard deviation: 0.05
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.92
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 6
Protein A protein: KIF1B
Protein A fragment: 517-526
Protein A site: 14-3-3_pThr522
Protein A modification: (TPO)522
Protein A sequence: REDGGTLGVF
Protein A construct: REDGG(pThr)LGVF (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.04
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.83
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: KIF1B
Protein A fragment: 517-526
Protein A site: 14-3-3_pThr522
Protein A modification: (TPO)522
Protein A sequence: REDGGTLGVF
Protein A construct: REDGG(pThr)LGVF (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.07
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.67
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 8
Protein A protein: KIF1B
Protein A fragment: 887-896
Protein A site: 14-3-3_pSer892
Protein A modification: (SEP)892
Protein A sequence: FKLVGSSPIF
Protein A construct: FKLVG(pSer)SPIF (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.49
Holdup BI standard deviation: 0.22
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.48
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: 3.83
Experiment series 9
Protein A protein: KIF1B
Protein A fragment: 1482-1491
Protein A site: 14-3-3_pSer1487
Protein A modification: (SEP)1487
Protein A sequence: RPRGDSLILE
Protein A construct: RPRGD(pSer)LILE (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: -0.05
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 116
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.91
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 10
Protein A protein: KIF1B
Protein A fragment: 1482-1491
Protein A site: 14-3-3_pSer1487
Protein A modification: (SEP)1487
Protein A sequence: RPRGDSLILE
Protein A construct: RPRGD(pSer)LILE (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.02
Holdup BI standard deviation: 0.07
Immobilized partner concentration in holdup experiment (10-6M): 143
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.25
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 11
Protein A protein: KIF1B
Protein A fragment: 1052-1061
Protein A site: 14-3-3_pSer1057
Protein A modification: (SEP)1057
Protein A sequence: TRSGLSLEEL
Protein A construct: TRSGL(pSer)LEEL (crude synthetic peptide)
Protein B protein: YWHAE
Protein B fragment: 1-255
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3epsilon
Average holdup BI: 0.02
Holdup BI standard deviation: 0.09
Immobilized partner concentration in holdup experiment (10-6M): 143
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.