Details of experiments between O00499 and Q8IU81
Note that the results of all experiments are listed, regardless of the modification states of the fragments.
Experiment series 1
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 1-584
Protein B construct: endogenous protein (Jurkat)
Average holdup BI: 0.16
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): 1.4
Experiment method: nHU_Jurkat
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.06.29.
Experimental details: -log10(P Two-sided unpaired T-test (6 vs 3))
Number of measurements: 3
The affinity was below the detection threshold of the assay.
Experiment series 2
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 1-584
Protein B construct: endogenous protein (Jurkat)
Average holdup BI: 0.48
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): 3.93
Experiment method: nHU_Jurkat
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022.06.29.
Experimental details: -log10(P Two-sided unpaired T-test (6 vs 3))
Number of measurements: 3
Measured pKd: 4.96
Experiment series 3
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.18
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.06.29.
Number of measurements: 2
Measured pKd: 4.12
Experiment series 4
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: T532M
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.06.29.
Number of measurements: 2
Measured pKd: 3.85
Experiment series 5
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: D537V
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.02
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
The affinity was below the detection threshold of the assay.
Experiment series 6
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: F584S
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.02
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
The affinity was below the detection threshold of the assay.
Experiment series 7
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: P551L
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 6
Experiment method: HU (DAPF)
Details of affinity fitting: Bait concentration was adjusted to fit the profile to WT
Date of measurement: 2022.10.24.
Number of measurements: 2
Measured pKd: 4.33
Experiment series 8
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Q540H
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.23
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
Measured pKd: 4.24
Experiment series 9
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: T532M
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.15
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
Measured pKd: 4.02
Experiment series 10
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V566M
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.14
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
Measured pKd: 3.97
Experiment series 11
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V583I
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.05
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 9
Experiment method: HU (DAPF)
Details of affinity fitting: Bait concentration was adjusted to fit the profile to WT
Date of measurement: 2022.10.24.
Number of measurements: 2
The affinity was below the detection threshold of the assay.
Experiment series 12
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.14
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
Measured pKd: 3.99
Experiment series 13
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Y531S
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
The affinity was below the detection threshold of the assay.
Experiment series 14
Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: R581C
Protein A construct: avi(biotin)-his6-MBP-TEVsite-SH3
Protein B protein: IRF2BP1
Protein B fragment: 361-375
Protein B site: PxxP_1
Protein B sequence: ALCGPPPRAPSRNLA
Protein B construct: Biotinylated 15-mer peptide (crude)
Average holdup BI: 0.09
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 6
Experiment method: HU (DAPF)
Details of affinity fitting: Bait concentration was adjusted to fit the profile to WT
Date of measurement: 2022.10.24.
Number of measurements: 2
Measured pKd: 4.23