Binding assay of Calreticulin using isothermal titration calorimetry

 GST-CRT was dialyzed against buffer A at least three times and concentrated using 10 kDa Amicon Ultra Centrifugal Filter Unit. 2 mL of protein solution was prepared (the final concentration was adjusted 30 μM). Concentration of GST-CRT was determined by A280 as 1.06 (1 mg/mL).

 1mg of Glc₁Man₉GlcNAc-OC₃H₇ was dissolved 1mL of buffer A (500 μM). 250 μL of the Glc₁Man₉GlcNAc-OC₃H₇ solution was added 150 μL of buffer A (the final concentration was adjusted 300 μM).

 The protein solution and the oligosaccharide solution were degassed under vacuum. (Strength of vacuum needs to be adjusted based on the behavior of the samples under vacuum.)

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

 Removal all air from the filling syringe

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

 Remove bubbles in the sample cell and adjusted of the sample volume (1.4181 mL)

 Loading of the oligosaccharide solution (300 μL) into the Auto Pipette using plastic syringe.

 Carefully insert the Auto-Pipette into the sample cell and spinning 300 rpm at 25°C.

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

 Titration data were fitted via the one set of site method to determine binding stoichiometry (n), binding constant (Ka), and change in enthalpy of binding (ΔH) using Origin software (Microcal).