Calreticulin (CRT) is a soluble, molecular chaperone found in endoplasmic reticulum (ER) of eukaryotes. It recognizes mono-glucosylated high mannose type N-glycan (Glc1Man9~7GlcNAc2) on nascent polypeptide and promotes the folding of glycoproteins in ER (calnexin / CRT cycle). |
Category | Sugar binding proteins |
Protocol Name | Binding assay of Calreticulin using isothermal titration calorimetry |
Authors
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Matsuo, Ichiro
Department of Chemistry and Chemical Biology, Faculty of Engineering, Gunma University
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KeyWords |
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Reagents
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buffer A (10 mM MOPS, 5 mM CaCl2, 150 mM NaCl, pH 7.4.) |
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2 mL of 30 μM GST-CRT solution in buffer A. |
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400 μL of 300 μM oligosaccharide solution in buffer A. |
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Instruments
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VP-ITC calorimeter (Microcal Inc., Northampton, MA)
- VP-ITC Cell (sample cell)
- Auto-Pipette (injection syringe)
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Hamilton 2.5mL filling syringe |
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10 kD Amicon Ultra (10 K) Centrifugal Filter Unit |
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Methods |
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Preparation of the samples
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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). |
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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). |
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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.) |
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Running an ITC experiment
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Slowly draw a minimum 1.8 mL of the protein solution into the filling syringe. |
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Removal all air from the filling syringe |
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Insert the syringe into the sample cell and slowly inject the protein solution. |
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Remove bubbles in the sample cell and adjusted of the sample volume (1.4181 mL) |
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Loading of the oligosaccharide solution (300 μL) into the Auto Pipette using plastic syringe. |
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Carefully insert the Auto-Pipette into the sample cell and spinning 300 rpm at 25°C. |
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The oligosaccharide solution was added as 50 injection of 6 μL into the sample cell (an interval of 3 min between each injection). |
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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). |
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Figure & Legends |
Figure & Legends |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-07-30 11:48:03 |
- Ito, Y., Hagihara, S., Matsuo, I., and Totani, K. (2005) Structural approaches to the study of oligosaccharides in glycoprotein quality control. Curr. Opin. Struct. Biol. 15, 481–489 [PMID : 16154739]
- Matsuo, I., Wada, M., Manabe, S., Yamaguchi, Y., Otake, K., Kato, K., and Ito, Y. (2003) Synthesis of monoglucosylated high-mannose type dodecasaccharide, a putative ligand for molecular chaperone, Calnexin and Calreticurin. J. Am. Chem. Soc. 125, 3402–3403 [PMID : 12643681]
- Kapoor, M., Srinivas, H., Kandiah, E., Gemma, E., Ellgaard, L., Oscarson, S., Helenius, A., and Surolia, A. (2003) Interactions of substrate with Calreticulin, an endoplasmic reticulum chaperone. J. Biol. Chem. 278, 6194–6200 [PMID : 12464625]
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(2014).
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