Details of experiments between Q8TBC3 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: SHKBP1
Protein A fragment: 698-707
Protein A site: PBM
Protein A sequence: PKMKLNETSF
Protein A construct: Biotin-Ahx-PKMKLNETSF
Protein B protein: SCRIB
Protein B fragment: 999-1093
Protein B site: PDZ_3
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.28
Normalized holdup BI: 0.34
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: The peptide stock solution had solubility issues. The peptide concentration in the affinity conversion step may differ from reality causing a systematic error in reported affinities.
PUBMED ID of publication: 36115835
Number of measurements: 1
Measured pKd: 4.49
Experiment series 2
Protein A protein: SHKBP1
Protein A fragment: 698-707
Protein A site: PBM
Protein A sequence: PKMKLNETSF
Protein A construct: Biotin-Ahx-PKMKLNETSF
Protein B protein: SCRIB
Protein B fragment: 718-814
Protein B site: PDZ_1
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.21
Holdup BI standard deviation: 0.01
Normalized holdup BI: 0.26
Normalized holdup BI standard deviation: 0.01
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: The peptide stock solution had solubility issues. The peptide concentration in the affinity conversion step may differ from reality causing a systematic error in reported affinities.
PUBMED ID of publication: 36115835
Number of measurements: 2
Measured pKd: 4.31
Experiment series 3
Protein A protein: SHKBP1
Protein A fragment: 698-707
Protein A site: PBM
Protein A sequence: PKMKLNETSF
Protein A construct: Biotin-Ahx-PKMKLNETSF
Protein B protein: SCRIB
Protein B fragment: 858-949
Protein B site: PDZ_2
Protein B construct: his6-MBP-TEVsite-PDZ
Average holdup BI: 0.02
Normalized holdup BI: 0.03
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: The peptide stock solution had solubility issues. The peptide concentration in the affinity conversion step may differ from reality causing a systematic error in reported affinities.
PUBMED ID of publication: 36115835
Number of measurements: 1
The affinity was below the detection threshold of the assay.
Experiment series 4
Protein A protein: SHKBP1
Protein A fragment: 1-707
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: 0.9
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): 5.29
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: 5.97
Experiment series 5
Protein A protein: SHKBP1
Protein A fragment: 1-707
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: 0.84
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): 5.8
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: 5.72