Details of experiments between Q96BN8 and Q14160
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: OTULIN
Protein A fragment: 343-352
Protein A site: PBM
Protein A sequence: PVRVCEETSL
Protein A construct: biotin-ttds-PVRVCEETSL
Protein B protein: SCRIB
Protein B fragment: 999-1093
Protein B site: PDZ_3
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.78
Normalized holdup BI: 0.86
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: DAPF_holdup
Details of affinity fitting: fluorescein + mCherry internal standards + normalization based on CALIP_holdup data + reverse layout
PUBMED ID of publication: 36115835
Number of measurements: 1
Measured pKd: 5.64
Experiment series 2
Protein A protein: OTULIN
Protein A fragment: 343-352
Protein A site: PBM
Protein A sequence: PVRVCEETSL
Protein A construct: biotin-ttds-PVRVCEETSL
Protein B protein: SCRIB
Protein B fragment: 718-814
Protein B site: PDZ_1
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.76
Normalized holdup BI: 0.8
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: DAPF_holdup
Details of affinity fitting: fluorescein + mCherry internal standards + normalization based on CALIP_holdup data + reverse layout
PUBMED ID of publication: 36115835
Number of measurements: 1
Measured pKd: 5.44
Experiment series 3
Protein A protein: OTULIN
Protein A fragment: 343-352
Protein A site: PBM
Protein A sequence: PVRVCEETSL
Protein A construct: biotin-ttds-PVRVCEETSL
Protein B protein: SCRIB
Protein B fragment: 858-949
Protein B site: PDZ_2
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.36
Holdup BI standard deviation: 0.02
Normalized holdup BI: 0.45
Normalized holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: DAPF_holdup
Details of affinity fitting: fluorescein + mCherry internal standards + normalization based on CALIP_holdup data + reverse layout
PUBMED ID of publication: 36115835
Number of measurements: 2
Measured pKd: 4.7
Experiment series 4
Protein A protein: OTULIN
Protein A fragment: 343-352
Protein A site: PBM
Protein A sequence: PVRVCEETSL
Protein A construct: Biotin-ado-ado-PVRVCEETSL
Protein B protein: SCRIB
Protein B fragment: 999-1093
Protein B site: PDZ_3
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.64
Normalized holdup BI: 0.76
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: DAPF_holdup
Details of affinity fitting: fluorescein + mCherry internal standards + normalization based on CALIP_holdup data + reverse layout
Experimental details: Crude peptide synthesis product. The peptide concentration in the affinity conversion step may differ from reality. Not reported internal standards of holdup layout #6
PUBMED ID of publication: 36115835
Number of measurements: 1
Measured pKd: 5.32
Experiment series 5
Protein A protein: OTULIN
Protein A fragment: 343-352
Protein A site: PBM
Protein A sequence: PVRVCEETSL
Protein A construct: Biotin-ado-ado-PVRVCEETSL
Protein B protein: SCRIB
Protein B fragment: 718-814
Protein B site: PDZ_1
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.89
Normalized holdup BI: 0.89
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: DAPF_holdup
Details of affinity fitting: fluorescein + mCherry internal standards + normalization based on CALIP_holdup data + reverse layout
Experimental details: Crude peptide synthesis product. The peptide concentration in the affinity conversion step may differ from reality. Not reported internal standards of holdup layout #6
PUBMED ID of publication: 36115835
Number of measurements: 1
Measured pKd: 5.74
Experiment series 6
Protein A protein: OTULIN
Protein A fragment: 343-352
Protein A site: PBM
Protein A sequence: PVRVCEETSL
Protein A construct: Biotin-ado-ado-PVRVCEETSL
Protein B protein: SCRIB
Protein B fragment: 858-949
Protein B site: PDZ_2
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.38
Normalized holdup BI: 0.38
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: DAPF_holdup
Details of affinity fitting: fluorescein + mCherry internal standards + normalization based on CALIP_holdup data + reverse layout
Experimental details: Crude peptide synthesis product. The peptide concentration in the affinity conversion step may differ from reality. Not reported internal standards of holdup layout #6
PUBMED ID of publication: 36115835
Number of measurements: 1
Measured pKd: 4.57
Experiment series 7
Protein A protein: OTULIN
Protein A fragment: 1-352
Protein A construct: endogenous protein (Jurkat)
Protein B protein: SCRIB
Protein B fragment: 718-814
Protein B site: PDZ_1
Protein B construct: avi(biotin)-his6-MBP-TEVsite-PDZ
Average holdup BI: 0.84
Immobilized partner concentration in holdup experiment (10-6M): 10
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.74
Experiment method: nHU_Jurkat
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.08.03.
Experimental details: -log10(P Two-sided unpaired T-test (6 vs 3))
PUBMED ID of publication: 36542723
Number of measurements: 3
Measured pKd: 5.73
Experiment series 8
Protein A protein: OTULIN
Protein A fragment: 1-352
Protein A construct: endogenous protein (Jurkat)
Protein B protein: SCRIB
Protein B fragment: 858-949
Protein B site: PDZ_2
Protein B construct: avi(biotin)-his6-MBP-TEVsite-PDZ
Average holdup BI: 0.26
Immobilized partner concentration in holdup experiment (10-6M): 10
P value (usually -log10(P Two-sided unpaired T-test), but double check the experimental details): 4.41
Experiment method: nHU_Jurkat
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.08.03.
Experimental details: -log10(P Two-sided unpaired T-test (6 vs 3))
PUBMED ID of publication: 36542723
Number of measurements: 3
The affinity was below the detection threshold of the assay.
Experiment series 9
Protein A protein: OTULIN
Protein A fragment: 343-352
Protein A site: PBM
Protein A sequence: PVRVCEETSL
Protein A construct: biotin-ttds-PVRVCEETSL
Protein B protein: SCRIB
Protein B fragment: 1-1630
Protein B construct: endogenous protein (Jurkat)
Experiment method: nHU-WB_Jurkat
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.08.03.
PUBMED ID of publication: 36542723
Number of measurements: 3
Measured pKd: 6.22