Microcal VP-ITC instruments and a Microcal iTC200. These
ITC instruments have similar cell geometries, but the working
volume of the VP-ITC is sevenfold larger.
l
SPR: Measurements should be made with an instrument with
appropriate sensitivity for small molecule binding to proteins
(the analyte:ligand mass ratio is approximately 0.04). The instrument should be capable of accepting sensor chips amenable to
immobilization of ligand via free amines using NHS-ester chemistry. The data presented here were measured using a Biacore
T200 with Biacore S-Series CM5 (carboxymethyl dextran
coated) sensor chips. Samples should be prepared and measured
in plasticware recommended by the instrument manufacturer.
3 Methods
3.1 Preparation
of Protein and Ligand
Solutions
3.1.1 Preparation
of HEWL Solution
1. The HEWL solution should be prepared by raising the lyophilized powder in the standard assay buffer at approximately the
correct concentration (a typical stock concentration would be
0.5 mg/mL, corresponding to 35 μM).
2. This solution should be filtered through a 0.2-μm syringe filter
(with a polyvinylidene fluoride membrane) to remove any
larger particulate material.
3. The accurate concentration of the HEWL solution should then
be measured by absorbance spectrophotometry, using the standard assay buffer as a reference (see Note 8). The measured
absorbance should be corrected for any contributions from
scattering using the formula A 280
corrected
¼ A 280
measured
À 2*A 333 (see Note 9). The concentration should be
calculated from the corrected 280 nm absorbance using the
Beer–Lambert law and a molar extinction coefficient (ε 280 ) of
37,970 M
À1 cm
À1 calculated using ProtParam from the primary sequence (see Note 10).
4. The HEWL solution should be prepared fresh before experiments and can be kept at room temperature for a few hours
before use. We do not use the solution for more than 1 day,
preferring always to make a new stock.
Obtaining accurate concentration for the HEWL solution is
crucial for consistency and accuracy of the experiments, especially in
those experiments where the working concentration is on the same
order as the K d (fluorescence, ITC), such that concentration errors
have a significant effect on fitted parameters. Where possible, it is
best to measure the concentration of the experimental solution
directly to avoid concentration errors from dilution. For example,
a working concentration of 35 μM for an ITC experiment gives an
absorbance of 1.33 in a 1 cm pathlength cuvette, which can be
measured reliably.
54
Xiaochun Li-Blatter et al.
ITC instruments have similar cell geometries, but the working
volume of the VP-ITC is sevenfold larger.
l
SPR: Measurements should be made with an instrument with
appropriate sensitivity for small molecule binding to proteins
(the analyte:ligand mass ratio is approximately 0.04). The instrument should be capable of accepting sensor chips amenable to
immobilization of ligand via free amines using NHS-ester chemistry. The data presented here were measured using a Biacore
T200 with Biacore S-Series CM5 (carboxymethyl dextran
coated) sensor chips. Samples should be prepared and measured
in plasticware recommended by the instrument manufacturer.
3 Methods
3.1 Preparation
of Protein and Ligand
Solutions
3.1.1 Preparation
of HEWL Solution
1. The HEWL solution should be prepared by raising the lyophilized powder in the standard assay buffer at approximately the
correct concentration (a typical stock concentration would be
0.5 mg/mL, corresponding to 35 μM).
2. This solution should be filtered through a 0.2-μm syringe filter
(with a polyvinylidene fluoride membrane) to remove any
larger particulate material.
3. The accurate concentration of the HEWL solution should then
be measured by absorbance spectrophotometry, using the standard assay buffer as a reference (see Note 8). The measured
absorbance should be corrected for any contributions from
scattering using the formula A 280
corrected
¼ A 280
measured
À 2*A 333 (see Note 9). The concentration should be
calculated from the corrected 280 nm absorbance using the
Beer–Lambert law and a molar extinction coefficient (ε 280 ) of
37,970 M
À1 cm
À1 calculated using ProtParam from the primary sequence (see Note 10).
4. The HEWL solution should be prepared fresh before experiments and can be kept at room temperature for a few hours
before use. We do not use the solution for more than 1 day,
preferring always to make a new stock.
Obtaining accurate concentration for the HEWL solution is
crucial for consistency and accuracy of the experiments, especially in
those experiments where the working concentration is on the same
order as the K d (fluorescence, ITC), such that concentration errors
have a significant effect on fitted parameters. Where possible, it is
best to measure the concentration of the experimental solution
directly to avoid concentration errors from dilution. For example,
a working concentration of 35 μM for an ITC experiment gives an
absorbance of 1.33 in a 1 cm pathlength cuvette, which can be
measured reliably.
54
Xiaochun Li-Blatter et al.