Cells were lysed with buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 10 mM sodium fluoride, 20 mM 2-ME, 250 μM sodium orthovanadate and 1 mM phenylmethane sulfonyl fluoride, and incubated at 4°C for 1 h. The cell lysates were ultrasonicated and centrifuged at 12,000 × g for 10 min. Protein concentrations were determined with the bicinchoninic acid assay. Protein samples were separated by 8% SDS-PAGE and electroblotted onto nitrocellulose membranes (EMD Millipore). Following blocking with TBS and 5% non-fat dry milk for 2 h, the membrane was incubated overnight at 4°C with antibodies against dentin sialophosphoprotein (DSPP, rabbit anti-human polyclonal antibody, 1:1,000, bs-10316R, Bioss), bone morphogenetic protein (BMP)2 (rabbit anti-human polyclonal antibody, 1:1,000, bs-1012R, Bioss), dentin matrix acidic phosphoprotein 1 (DMP1, rabbit anti-human polyclonal antibody, 1:1,000, bs-12359R, Bioss) and runt-related transcription factor (RUNX)2 (rabbit anti-human polyclonal antibody, 1:1,000, bs-1134R, Bioss), followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 45 min at room temperature. After each incubation, the membrane was thoroughly washed with TBS-Tween-20. Subsequently, a coloring reaction was performed with ECL and the ratio was quantified using a GelDoc XR System (Bio-Rad Laboratories, Inc.).
Bs 10316r
Manufactured by Bioss Antibodies
Sourced in China, United States
The Bs-10316R is a lab equipment product offered by Bioss Antibodies. It serves as a core function in laboratory settings, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information on the intended use of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Gel doc xr system, by Bio-Rad (1 mentions)
Bs 1134r, by Bioss Antibodies (1 mentions)
Bs 12359r, by Bioss Antibodies (1 mentions)
Bs 1012r, by Bioss Antibodies (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
P36962, by Thermo Fisher Scientific (1 mentions)
F9006, by Merck Group (1 mentions)
Dmi6b, by Leica (1 mentions)
Goat anti mouse fitc, by Merck Group (1 mentions)
3 protocols using bs 10316r
1
Western Blot Analysis of Dental Proteins
2
Western Blot Analysis of Osteogenic Markers
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To detect proteins of osteo/odontogenic differentiation genes, DPSCs were cultured in mineralization-inducing medium for 2 weeks, and then western blot analysis was performed as previously described [34 (link)]. Primary antibodies were obtained from commercial sources as follows: polyclonal antibody against bone sialoprotein (BSP) (Cat. No. bs-2668R, Bioss, Beijing, China); monoclonal antibody against osteocalcin (OCN) (Cat. No. ab133612, Abcam, Cambridge, UK); polyclonal antibody against dentin sialophosphoprotein (DSPP) (Cat. No. bs-10316R Bioss, Beijing, China); polyclonal antibody against beta-actin (β-actin) (Cat. No. AC026, Abclonal, Wuhan, China).
3
Immunocytochemical Analysis of DPSC Markers
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The immunocytochemical analysis was performed by the markers dentin matrix protein 1 (anti-DMP1, bs-12359R, Bioss, USA), dentin sialophosphoprotein (anti-DSPP, bs-10316R, Bioss, USA), and nestin (anti-nestin, bs-0008R, Bioss, USA). Primary antibodies were diluted to 1:200 for all primer antibodies. DPSCs were seeded on 8-well chamber slides (10,000 cells/well). After the differentiation process, cells were fixed with methanol. For double-labeling, cells were incubated with the first primary antibody (anti-nestin) overnight at 4°C, followed by its specific secondary antibody (goat anti-mouse FITC, 1:1000, F9006, Sigma-Aldrich, USA). Thereafter, cells were incubated with one of the second primary antibodies (anti-DMP1 or anti-DSPP) and its specific secondary antibody (donkey anti-rabbit Alexa Fluor 568, 1:1000, A10042, Invitrogen, Carlsbad, CA, USA). Sections were mounted with fluorescence mounting medium containing DAPI (P36962, Thermo Fisher Scientific, Waltham, MA, USA). Stained cells were visualized using a light microscope with fluorescence attachment (DMI6B, Leica, Wetzlar, Germany).
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