Protein Extraction from Bone

Protein extraction from bone
Extractsof intramembranous bone (flat bones: calvariae and scapulae)and endochondral bone (long bones: femurandtibia)from rats were prepared essentially as described byGoldberg et al.((35))In brief, 3-month-old male CD rats (Charles RiverLaboratories, St. Constant, QC, Canada) were killed withcarbon dioxide and their flat bonesand longbones were excised and flash-frozen with liquid nitrogen.Bones were crushed into a fine powder witha biopulverizer (Biospec Products, Bartlesville, OK, USA) underliquid nitrogen, and the bone powder was subjectedto two, 24-h extractions at 4°Cwith 4 M of guanidine-HCl in 50 mMof Tris-HCl, pH 7.4, containing protease inhibitors (1mM of phenylmethylsulfonylfluoride, 1 mg/ml of benzamidine, and1 mg/ml of leupeptin [allfrom Sigma]).After spinning (1000gfor 15 minutes), the pellet was washed twicewith 50 mM of Tris-HCl, pH 7.4, andthe preparation was subjected to two, 24-h extractionswith 0.5 M of EDTA in 50 mMof Tris-HCl, pH 7.4, at 4°C.EDTA extraction was followed by two additional extractionswith 4 M of guanidine-HCl as described previously.Extracts were named G1-extract, E-extract, and G2-extract, respectively,as conventionally used for this extraction scheme.((35,36))All extracts were concentrated using Centricon Plus-20 PL-10concentrators with a molecular weight cut-off of 10kDa (Millipore, Bedford, MA, USA) and buffers werechanged to 8 M of urea (for G1-and G2-extracts) and to 5 mM of NH4HCO3,pH 8 (for E-extract). Protein concentrations were determinedusing the bicinchoninic acid (BCA) protein assay (Pierce,Rockford, IL, USA). Success of the extraction wasconfirmed by SDS-PAGE and silver staining.((37))

extraction was performed using a modification of apreviously described procedure.((3))In brief, demineralization of bony tissues (vertebra andjaw) and calcified cartilage (branchial arches) was donewith a 10-fold excess of 10%formic acid (v/w) at 4°Cfor 4 h with continuous stirring. The resultingacid extracts were dialyzed at 4°Cagainst 50 mM HCl using a 3500 molecularweight cut-off tubing (SpectraPor 3; Spectrum, Gardena, CA,USA) with four changes of the medium over2 days to remove all dissolved mineral. Theentire dialyzed extract was freeze-dried, and two identicalsamples (approximately 30 µgof total protein) from each extract were loadedonto two adjacent lanes and analyzed by SDS-PAGE.After electrophoresis, the two identical lanes were separatedby cutting the gel in two, and theprotein profile in each half was revealed bystaining either with Coomassie Brilliant blue or witha Gla-specific color reaction((23))as described below.

Preparation of tissues for proteinextraction and immunoblotting
Fifteen-day-oldmice were killed by cervical dislocation and wholejaws were dissected under stereo-microscope. The incisors andmolars were taken out carefully to avoid contaminationby other tissues. Teeth were homogenized and incubatedon ice for 30 minutes in 1lysis buffer (50 mM of Tris-HCl, pH 7.4,250 mM of NaCl, 2 mM of EDTA,2 µg/mlof aprotinin, 1 mM dithiothreitol [DTT],1 mM of phenyl methyl sulfonyl fluoride, 1mM of NaF, 0.5 µg/mlof leupeptin, and 1%NP-40). The lysate was centrifuged at 10,000 rpmat 4°Cfor 30 minutes.