Protein extraction from bone
Extracts of intramembranous bone (flat bones: calvariae and scapulae) and
endochondral bone (long bones: femur and tibia) from rats were prepared essentially as
described by Goldberg et al.((35)) In brief, 3-month-old male CD rats (Charles River
Laboratories, St. Constant, QC, Canada) were killed with carbon dioxide and their flat
bones and long bones were excised and flash-frozen with liquid nitrogen. Bones were
crushed into a fine powder with a biopulverizer (Biospec Products, Bartlesville, OK,
USA) under liquid nitrogen, and the bone powder was subjected to two, 24-h extractions
at 4°C with 4 M of guanidine-HCl in 50 mM of Tris-HCl, pH 7.4, containing protease
inhibitors (1 mM of phenylmethylsulfonylfluoride, 1 mg/ml of benzamidine, and 1 mg/ml
of leupeptin [all from Sigma]). After spinning (1000g for 15 minutes), the pellet was
washed twice with 50 mM of Tris-HCl, pH 7.4, and the preparation was subjected to two,
24-h extractions with 0.5 M of EDTA in 50 mM of Tris-HCl, pH 7.4, at 4°C. EDTA
extraction was followed by two additional extractions with 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-10 concentrators with a molecular weight cutoff of 10 kDa (Millipore, Bedford, MA, USA) and buffers were changed to 8 M of urea
(for G1- and G2-extracts) and to 5 mM of NH4HCO3, pH 8 (for E-extract). Protein
concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce,
Rockford, IL, USA). Success of the extraction was confirmed by SDS-PAGE and silver
staining.((37))
extraction was performed using a modification of a previously described procedure.((3))
In brief, demineralization of bony tissues (vertebra and jaw) and calcified cartilage
(branchial arches) was done with a 10-fold excess of 10% formic acid (v/w) at 4°C for 4 h
with continuous stirring. The resulting acid extracts were dialyzed at 4°C against 50 mM
HCl using a 3500 molecular weight cut-off tubing (SpectraPor 3; Spectrum, Gardena,
CA, USA) with four changes of the medium over 2 days to remove all dissolved mineral.
The entire dialyzed extract was freeze-dried, and two identical samples (approximately
30 µg of total protein) from each extract were loaded onto two adjacent lanes and
analyzed by SDS-PAGE. After electrophoresis, the two identical lanes were separated by
cutting the gel in two, and the protein profile in each half was revealed by staining either
with Coomassie Brilliant blue or with a Gla-specific color reaction((23)) as described
below.
Preparation of tissues for protein extraction and immunoblotting
Fifteen-day-old mice were killed by cervical dislocation and whole jaws were dissected
under stereo-microscope. The incisors and molars were taken out carefully to avoid
contamination by other tissues. Teeth were homogenized and incubated on ice for 30
minutes in 1 lysis buffer (50 mM of Tris-HCl, pH 7.4, 250 mM of NaCl, 2 mM of
EDTA, 2 µg/ml of aprotinin, 1 mM dithiothreitol [DTT], 1 mM of phenyl methyl sulfonyl
fluoride, 1 mM of NaF, 0.5 µg/ml of leupeptin, and 1% NP-40). The lysate was
centrifuged at 10,000 rpm at 4°C for 30 minutes.