Mouse uncarboxylated OCN was purified from BL21 transformed with pGEX2TK-mOCN as previously described (Lee et al., 2007 (link); Ferron et al., 2010a (link); Oury et al., 2011 (link), 2013 (link); Mera et al., 2016 (link)). In brief, GST-OCN fusion protein was bacterially produced in BL21 pLyS transformed with pGEX2TK-mOCN after induction with IPTG. Cells were collected in lysis buffer (PBS 1×, 10 mM Tris, pH 7.2, 2 mM EDTA, 1% Triton, and 1× protease and phosphatase inhibitor cocktail; 78443; Thermo Fisher Scientific). Following four freeze-thaw cycles and sonication, lysates were cleared by centrifugation. The supernatant was incubated with glutathione-Sepharose 4B (17075601; GE) for 4 h at 4°C. Following six washes with washing buffer (PBS 1× and 1% Triton) and with PBS 1×, OCN was then cleaved out from the GST moiety by using thrombin (27-0846-01; GE). Four fractions were collected, and each of them was incubated with benzamidine Sepharose (17-5123-10; GE) for 30 min at room temperature to remove thrombin.
Protocol detail
Purification of Uncarboxylated Osteocalcin
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Benzamidine
Buffer
Cells
Centrifugation
Edta
Ferron
Freeze
Glutathione
Iptg
Mouse
Phosphatase
Protease
Protein
Sepharose
Sepharose 4b
Thrombin
Tris
Corresponding Organization :
Other organizations : Columbia University Irving Medical Center, Inserm, Université Paris Cité, Sorbonne Paris Cité, Délégation Paris 5, Institut Necker Enfants Malades, Grenoble Institute of Neurosciences, Université Grenoble Alpes, Howard Hughes Medical Institute, Columbia University, New York Psychoanalytic Society and Institute, Centre Hospitalier Universitaire de Grenoble, Yale University
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Variable analysis
independent variables
- Transformation of BL21 cells with pGEX2TK-mOCN plasmid
- Induction of GST-OCN fusion protein expression with IPTG
- Purification of uncarboxylated mouse OCN protein
- Lysis buffer composition (PBS 1x, 10 mM Tris, pH 7.2, 2 mM EDTA, 1% Triton, and 1x protease and phosphatase inhibitor cocktail)
- Freeze-thaw cycles and sonication of cell lysates
- Incubation of supernatant with glutathione-Sepharose 4B for 4 hours at 4°C
- Washing steps with washing buffer (PBS 1x and 1% Triton) and PBS 1x
- Cleavage of OCN from GST moiety using thrombin
- Incubation of fractions with benzamidine Sepharose for 30 minutes at room temperature to remove thrombin
- Not explicitly mentioned
- Not explicitly mentioned
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