Details of experiments between O00499 and Q8IY57
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: YAF2
Protein B fragment: 1-180
Protein B construct: endogenous protein (Jurkat)

Average holdup BI: 0.59
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): 5.72
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: 5.15

Experiment series 2

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

Protein B protein: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.49
Holdup BI standard deviation: 0
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: 4.78

Experiment series 3

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.51
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: 4.81

Experiment series 4

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.51
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: 4.81

Experiment series 5

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.04
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: 2023/01/17
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: V583I
Protein A construct: his6-MBP-TEVsite-SH3

Protein B protein: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.33
Holdup BI standard deviation: 0.01
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
Measured pKd: 4.62

Experiment series 7

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.26
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
Measured pKd: 4.68

Experiment series 8

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.01
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
The affinity was below the detection threshold of the assay.

Experiment series 9

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.41
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: 4.62

Experiment series 10

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.3
Holdup BI standard deviation: 0
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
Measured pKd: 4.68

Experiment series 11

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: YAF2
Protein B fragment: 3-17
Protein B site: PxxP_1
Protein B sequence: DKKSPTRPKRQPKPS
Protein B construct: Bio Ado Ado-DKKSPTRPKRQPKPS-CONH2

Average holdup BI: 0.5
Holdup BI standard deviation: 0
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: 4.8