Details of experiments between Q6R327 and P27348
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: RICTOR
Protein A fragment: 1279-1288
Protein A site: 14-3-3_pSer1284
Protein A modification: (SEP)1284
Protein A sequence: LSKSNSVSLV
Protein A construct: LSKSN(pSer)VSLV (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.37
Holdup BI standard deviation: 0.04
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.95
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 2
Protein A protein: RICTOR
Protein A fragment: 1214-1223
Protein A site: 14-3-3_pSer1219
Protein A modification: (SEP)1219
Protein A sequence: KIRSQSFNTD
Protein A construct: KIRSQ(pSer)FNTD (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.53
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.47
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: 3.93
Experiment series 3
Protein A protein: RICTOR
Protein A fragment: 1297-1306
Protein A site: 14-3-3_pSer1302
Protein A modification: (SEP)1302
Protein A sequence: PRRAQSLKAP
Protein A construct: PRRAQ(pSer)LKAP (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.7
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 5.04
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: 4.26
Experiment series 4
Protein A protein: RICTOR
Protein A fragment: 1128-1137
Protein A site: 14-3-3_pThr1133
Protein A modification: (TPO)1133
Protein A sequence: NRRIRTLTEP
Protein A construct: NRRIR(pThr)LTEP (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.28
Holdup BI standard deviation: 0.08
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 3.41
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
Measured pKd: 3.48
Experiment series 5
Protein A protein: RICTOR
Protein A fragment: 1172-1181
Protein A site: 14-3-3_pSer1177
Protein A modification: (SEP)1177
Protein A sequence: TGSTPSIGEN
Protein A construct: TGSTP(pSer)IGEN (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: -0.2
Holdup BI standard deviation: 0.1
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 2.53
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: RICTOR
Protein A fragment: 1380-1389
Protein A site: 14-3-3_pSer1385
Protein A modification: (SEP)1385
Protein A sequence: FMKALSYASL
Protein A construct: FMKAL(pSer)YASL (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.41
Holdup BI standard deviation: 0.21
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.54
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
Measured pKd: 3.72
Experiment series 7
Protein A protein: RICTOR
Protein A fragment: 1130-1139
Protein A site: 14-3-3_pThr1135
Protein A modification: (TPO)1135
Protein A sequence: RIRTLTEPSV
Protein A construct: RIRTL(pThr)EPSV (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.27
Holdup BI standard deviation: 0.19
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.49
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: 3.46
Experiment series 8
Protein A protein: RICTOR
Protein A fragment: 1133-1142
Protein A site: 14-3-3_pSer1138
Protein A modification: (SEP)1138
Protein A sequence: TLTEPSVDFN
Protein A construct: TLTEP(pSer)VDFN (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: -0.1
Holdup BI standard deviation: 0.1
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 1.42
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: RICTOR
Protein A fragment: 1327-1336
Protein A site: 14-3-3_pThr1332
Protein A modification: (TPO)1332
Protein A sequence: AFGYATLKRL
Protein A construct: AFGYA(pThr)LKRL (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: -0.25
Holdup BI standard deviation: 0.43
Immobilized partner concentration in holdup experiment (10-6M): 130
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 0.68
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: RICTOR
Protein A fragment: 1383-1392
Protein A site: 14-3-3_pSer1388
Protein A modification: (SEP)1388
Protein A sequence: ALSYASLDKE
Protein A construct: ALSYA(pSer)LDKE (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.03
Holdup BI standard deviation: 0.06
Immobilized partner concentration in holdup experiment (10-6M): 130
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 11
Protein A protein: RICTOR
Protein A fragment: 378-387
Protein A site: 14-3-3_pSer383
Protein A modification: (SEP)383
Protein A sequence: PHRARSRPDL
Protein A construct: PHRAR(pSer)RPDL (crude synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Average holdup BI: 0.04
Holdup BI standard deviation: 0.21
Immobilized partner concentration in holdup experiment (10-6M): 130
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 12
Protein A protein: RICTOR
Protein A fragment: 1214-1223
Protein A site: 14-3-3_pSer1219
Protein A modification: (SEP)1219
Protein A sequence: KIRSQSFNTD
Protein A construct: biotin-ttds-KIRSQ(pSer)FNTD (HPLC-purified synthetic peptide)
Protein B protein: YWHAQ
Protein B fragment: 1-245
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3tau
Measured dissociation constant (10-6M): 109
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: 2022.02.08
Number of measurements: 3
Measured pKd: 3.96