Details of experiments between Q14978 and O00499
Note that the results of all experiments are listed, regardless of the modification states of the fragments.


Experiment series 1

Protein A protein: NOLC1
Protein A fragment: 1-699
Protein A construct: endogenous protein (Jurkat)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.06
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): 3.59
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: NOLC1
Protein A fragment: 1-699
Protein A construct: endogenous protein (Jurkat)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.72
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): 1.46
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 3

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

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: 3.83

Experiment series 4

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: T532M
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.07
Holdup BI standard deviation: 0.03
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: 3.66

Experiment series 5

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: D537V
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0
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: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: F584S
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: -0.01
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 7

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: P551L
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.03
Holdup BI standard deviation: 0.05
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
The affinity was below the detection threshold of the assay.

Experiment series 8

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: Q540H
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.07
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: 3.62

Experiment series 9

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: T532M
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.05
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 10

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: V566M
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.04
Holdup BI standard deviation: 0
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 11

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: V583I
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.03
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: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.06
Holdup BI standard deviation: 0
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: 3.53

Experiment series 13

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: Y531S
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.21
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: 4.19

Experiment series 14

Protein A protein: NOLC1
Protein A fragment: 193-207
Protein A site: PxxP_1
Protein A sequence: KAQTKAPPKPARAAP
Protein A construct: Biotinylated 15-mer peptide (crude)

Protein B protein: BIN1
Protein B fragment: 513-593
Protein B site: SH3
Protein B modification: R581C
Protein B construct: avi(biotin)-his6-MBP-TEVsite-SH3

Average holdup BI: 0.02
Holdup BI standard deviation: 0.03
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
The affinity was below the detection threshold of the assay.