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Purification and Characterization of Calreticulin: a Ca2+-Binding Chaperone from Sheep Kidney

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Abstract

Calreticulin (CRT) is a molecular chaperone with a molecular mass of 46 kDa present in the endoplasmic reticulum (ER). This protein is primarily involved in the regulation of intracellular Ca2+ homeostasis and Ca2+ storage in the ER. CRT also plays a significant role in autoimmunity and cancer. This protein contains three distinct structural domains with specialized functions. Here, we are reporting a simple procedure for the purification of CRT from mammalian kidney. To isolate CRT,  sheep kidney was crushed and kept for 12 h in the extraction buffer. The lysate was centrifuged, and supernatant was precipitated by ammonium sulphate. The precipitate of 90 % ammonium sulphate was extensively dialyzed and loaded on DEAE-Hi-Trap FF and Mono Q chromatography columns. The purity of CRT was confirmed by SDS-PAGE. Finally, the protein was identified by matrix-assisted laser desorption/ionization time of flight. The purified protein was further characterized for secondary structural elements using the far-UV circular dichroism measurements. Our purification procedure is fast and simple with high yield.

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Abbreviations

CRT:

Calreticulin

ER:

Endoplasmic reticulum

DEAE:

Diethylaminoethanol

PDI:

Protein disulphide isomerase

EDTA:

Ethylenediamine tetraacetate

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Acknowledgments

We thank the Director of Slaughter house, Ghazipur, Delhi, for arranging sheep kidney tissues. Md. Aasif thanks the Indian Council of Medical Research for the award of fellowship. F.A. thanks CSIR and DST for the financial support. We also thank Dr. Souvik Maiti, Institute of Genomics and Integrative Biology (India), for his help with CD measurements.

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India

    Mohammad Aasif Dar,  Wahiduzzaman, Asimul Islam, Md. Imtaiyaz Hassan & Faizan Ahmad

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  1. Mohammad Aasif Dar
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  2. Wahiduzzaman
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  3. Asimul Islam
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  4. Md. Imtaiyaz Hassan
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  5. Faizan Ahmad
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Correspondence to Faizan Ahmad.

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Dar, M.A., Wahiduzzaman, Islam, A. et al. Purification and Characterization of Calreticulin: a Ca2+-Binding Chaperone from Sheep Kidney. Appl Biochem Biotechnol 174, 1771–1783 (2014). https://doi.org/10.1007/s12010-014-1150-5

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