Details of experiments between Q96BN8 and Q12959
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: DLG1
Protein B fragment: 314-412
Protein B site: PDZ_2
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.33
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
PUBMED ID of publication: 36115835
Number of measurements: 1
Measured pKd: 4.56
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: DLG1
Protein B fragment: 221-314
Protein B site: PDZ_1
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.23
Normalized holdup BI: 0.28
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: 4.35
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: DLG1
Protein B fragment: 457-570
Protein B site: PDZ_3
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.16
Normalized holdup BI: 0.19
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: 4.14
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: DLG1
Protein B fragment: 221-314
Protein B site: PDZ_1
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.71
Normalized holdup BI: 0.84
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: 5.54
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: DLG1
Protein B fragment: 314-412
Protein B site: PDZ_2
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.33
Normalized holdup BI: 0.37
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: 4.55
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: DLG1
Protein B fragment: 457-570
Protein B site: PDZ_3
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.09
Normalized holdup BI: 0.13
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: 3.94
Experiment series 7
Protein A protein: OTULIN
Protein A fragment: 1-352
Protein A construct: endogenous protein (Jurkat)
Protein B protein: DLG1
Protein B fragment: 221-314
Protein B site: PDZ_1
Protein B construct: avi(biotin)-his6-MBP-TEVsite-PDZ
Average holdup BI: 0.05
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): 0.56
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 8
Protein A protein: OTULIN
Protein A fragment: 1-352
Protein A construct: endogenous protein (Jurkat)
Protein B protein: DLG1
Protein B fragment: 314-412
Protein B site: PDZ_2
Protein B construct: avi(biotin)-his6-MBP-TEVsite-PDZ
Average holdup BI: 0.29
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): 4.47
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: 4.61