title: Purification and characterization of calreticulin: a Ca²⁺-binding chaperone from sheep kidney.
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 Ca(2+) homeostasis and Ca(2+) 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.
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India.
authors.forename: Mohammad Aasif
authors.initials: MA
authors.lastname: Dar
authors.lastname: Wahiduzzaman
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D037282:Calreticulin / Q000737:chemistry* / Q000302:isolation & purification*; D005591:Chemical Fractionation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D007668:Kidney / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry / Q000302:isolation & purification; D012756:Sheep
chemical_list: D002135:Calcium-Binding Proteins; D037282:Calreticulin; D018832:Molecular Chaperones