Details of experiments between O00499 and Q6PJT7
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: ZC3H14
Protein B fragment: 1-736
Protein B construct: endogenous protein (Jurkat)

Average holdup BI: 0.39
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): 5.68
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: 4.8

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: ZC3H14
Protein B fragment: 1-736
Protein B construct: endogenous protein (Jurkat)

Average holdup BI: 0.51
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): 3.13
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: 5.02

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.13
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: 3.92

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.08
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: 3.7

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

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: 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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: -0.04
Holdup BI standard deviation: 0.08
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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.04
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
The affinity was below the detection threshold of the assay.

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.08
Holdup BI standard deviation: 0.06
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: 3.7

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.07
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: 3.63

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.06
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: 3.58

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.01
Holdup BI standard deviation: 0.02
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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
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): 18
Experiment method: HU (DAPF)
Date of measurement: 2022.10.24.
Number of measurements: 2
Measured pKd: 3.76

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.21
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: 4.2

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: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Biotinylated 15-mer peptide (crude)

Average holdup BI: 0.03
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
The affinity was below the detection threshold of the assay.

Experiment series 15

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0.08
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/16
Number of measurements: 2
Measured pKd: 3.72

Experiment series 16

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: T532M
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0.09
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/16
Number of measurements: 2
Measured pKd: 3.75

Experiment series 17

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Q540H
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0.09
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 2
Measured pKd: 3.74

Experiment series 18

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: D537V
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: -0.02
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 2
The affinity was below the detection threshold of the assay.

Experiment series 19

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V583I
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0.03
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 13
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 2
The affinity was below the detection threshold of the assay.

Experiment series 20

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: P551L
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0.04
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 8
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 2
The affinity was below the detection threshold of the assay.

Experiment series 21

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: F584S
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: -0.01
Holdup BI standard deviation: 0.03
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/17
Number of measurements: 2
The affinity was below the detection threshold of the assay.

Experiment series 22

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: Y531S
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0.23
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/18
Number of measurements: 2
Measured pKd: 4.25

Experiment series 23

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: R581C
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0
Holdup BI standard deviation: 0.02
Immobilized partner concentration in holdup experiment (10-6M): 10
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/18
Number of measurements: 2
The affinity was below the detection threshold of the assay.

Experiment series 24

Protein A protein: BIN1
Protein A fragment: 513-593
Protein A site: SH3
Protein A modification: V566M
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: ZC3H14
Protein B fragment: 329-343
Protein B site: PxxP_1
Protein B sequence: SSSVSVPAKPERRPS
Protein B construct: Bio Ado Ado-SSSVSVPAKPERRPS-CONH2

Average holdup BI: 0.1
Holdup BI standard deviation: 0.01
Immobilized partner concentration in holdup experiment (10-6M): 18
Experiment method: HU (DAPF)
Details of affinity fitting: Quadratic binding equation
Date of measurement: 2023/01/18
Number of measurements: 2
Measured pKd: 3.78