Details of experiments between Q53ET0 and Q04917
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: CRTC2
Protein A fragment: 65-74
Protein A site: 14-3-3_pSer70
Protein A modification: (SEP)70
Protein A sequence: SHYGGSLPNV
Protein A construct: SHYGG(pSer)LPNV (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.81
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): 6.34
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: 4.52
Experiment series 2
Protein A protein: CRTC2
Protein A fragment: 122-131
Protein A site: 14-3-3_pSer127
Protein A modification: (SEP)127
Protein A sequence: TRHIDSSPYS
Protein A construct: TRHID(pSer)SPYS (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.43
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): 5.16
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: 3.77
Experiment series 3
Protein A protein: CRTC2
Protein A fragment: 166-175
Protein A site: 14-3-3_pSer171
Protein A modification: (SEP)171
Protein A sequence: LNRTSSDSAL
Protein A construct: LNRTS(pSer)DSAL (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.49
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): 4.27
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: 3.86
Experiment series 4
Protein A protein: CRTC2
Protein A fragment: 159-168
Protein A site: 14-3-3_pSer164
Protein A modification: (SEP)164
Protein A sequence: LFRLPSALNR
Protein A construct: LFRLP(pSer)ALNR (crude synthetic peptide)
Protein B protein: YWHAH
Protein B fragment: 1-246
Protein B site: 14-3-3
Protein B construct: avi-his6-MBP-TEVsite-14-3-3eta
Average holdup BI: 0.13
Holdup BI standard deviation: 0.56
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): 0.19
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.