Binding assay of calreticulin using isothermal titration calorimetry

Buffer A (10 mM of MOPS, 5 mM of CaCl₂, and 150 mM of NaCl, pH 7.4.)

2 mL of 30 μM of GST-CRT solution in buffer A

400 μL of 300 μM of oligosaccharide solution in buffer A

VP-ITC calorimeter (Malvern Panalytical Ltd., Worcestershire, UK) (Note 1).

a.

VP-ITC Cell (sample cell)

b.

Auto Pipette (injection syringe)

Hamilton 2.5-mL filling syringe

Plastic syringe (1 mL)

UV-visible spectrometer

Refrigerated centrifuge

10 kDa Amicon Ultra (10 K) Centrifugal Filter Unit

Protocol for the preparation of protein sample

a.

GST-CRT was dialyzed against buffer A at least three times and concentrated using 10 kDa Amicon Ultra Centrifugal Filter Unit.

b.

Protein concentration of GST-CRT was determined by A280 as 1.06 (1 mg/mL).

c.

2 mL of protein solution was prepared (the final concentration was adjusted at 30 μM) (Note 2).

d.

The protein solution was degassed under vacuum (strength of vacuum needs to be adjusted based on the behavior of the samples under vacuum).

Protocol for the preparation of oligosaccharide sample

a.

1 mg of Glc₁Man₉GlcNAc₂-OC₃H₇ was dissolved 1 mL of buffer A (500 μM).

b.

250 μL of the Glc₁Man₉GlcNAc₂-OC₃H₇ solution was added to 150 μL of buffer A (the final concentration was adjusted at 300 μM) (Note 3).

c.

The oligosaccharide solution was degassed under vacuum (strength of vacuum needs to be adjusted based on the behavior of the samples under vacuum).

Performing an experiment and data analysis

a.

Slowly draw a minimum 1.8 mL of the protein solution into the filling syringe.

b.

Remove all air from the filling syringe.

c.

Insert the syringe into the sample cell and slowly inject the protein solution.

d.

Remove bubbles in the sample cell and adjust the sample volume to 1.4181 mL (Note 4).

e.

Load the oligosaccharide solution (300 μL) into the Auto Pipette using a plastic syringe.

f.

Carefully insert the Auto Pipette into the sample cell with a spinning rate of 300 rpm at 25°C.

g.

The oligosaccharide solution was added as 50 injections of 6 μL into the sample cell (an interval of 3 min between each injection) (Note 5).

h.

Titration data as shown in Figure 1 were fitted using the one set of site method to determine binding stoichiometry (n), binding constant (KA) (Figure 2), and change in enthalpy of binding (ΔH) using Origin software (MicroCal).

The VP-ITC is not currently for sale, and the current type MicroCal PEAQ-ITC is for sale by Malvern Panalytical Ltd. The PEAQ-ITC has a cell capacity of 200 µL (required capacity of 280 µL) and a syringe capacity of 40 µL, which can significantly reduce sample consumption compared to VP-ITC. Additionally, degassing of sample solution is usually not required, and filling of ligand solution is automated, eliminating the need for complex operations.

A favorable concentration of lectin depends on KA, n, and ΔH (heat change in cal/mole). The unitless constant, c, which is derived by Wiseman et al. (6), is useful for experimental design.

• c = n × [P] × KA
• When c ranges from 5 to 250, a sigmoidal thermogram will be obtained, which provides accurate estimates of KA.

If a glycan sample that cannot be weighed on a balance, it should be quantified by the phenol–sulfuric acid method (7) using concentrated sulfuric acid (Sigma-Aldrich) and 5% aqueous phenol solution, which is prepared by adding 5 g of redistilled reagent grade phenol (Sigma-Aldrich) to 95 g of ultrapure water.

Dust, other particulates, and air bubbles can cause artifacts in the baseline of the ITC thermogram. The sample should be centrifuged in microcentrifuge tubes to remove pellet particles.

Control experiments should be conducted by injecting the glycan solution into the buffer.