Details of experiments between the fragments O00499~513-593 and Q8IZ21~286-300
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: his6-MBP-TEVsite-SH3
Protein B protein: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.49
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: 4.78
Experiment series 2
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.49
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: 4.78
Experiment series 3
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.55
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
Measured pKd: 4.88
Experiment series 4
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.11
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
Measured pKd: 3.83
Experiment series 5
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.3
Holdup BI standard deviation: 0
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: 4.57
Experiment series 6
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.25
Holdup BI standard deviation: 0
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: 4.67
Experiment series 7
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: -0.02
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 8
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.02
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
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: R581C
Protein A construct: his6-MBP-TEVsite-SH3
Protein B protein: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.27
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: 4.62
Experiment series 10
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: PHACTR4
Protein B fragment: 286-300
Protein B site: PxxP_4
Protein B sequence: LLSKPSPPLPPKRGI
Protein B construct: Bio Ado Ado-LLSKPSPPLPPKRGI-CONH2
Average holdup BI: 0.51
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: 4.82