Cell extracts containing 40 μg of total protein were separated by electrophoresis on sodium dodecyl sulphate polyacrylamide gels, and subsequently transferred to nitrocellulose membranes. After transfer, the membranes were blocked with PBS containing 5% non‐fat milk for 1 hour at room temperature, and then incubated at 4°C overnight with primary antibodies against Total AKT (T‐AKT, Boster, Wuhan, China), Phospho‐AKT (P‐AKT; Cell Signaling Technology), T‐GSK‐3β (Cell Signaling Technology), Phospho‐GSK‐3β (Cell Signaling Technology), β‐catenin (Cell Signaling Technology), Cyclin D1 (Abcam, Cambridge, MA, USA), Runt‐related transcription factor 2 (RUNX2; Boster), Bone morphogenic protein‐2 (BMP2; Boster) and Bone sialoprotein (BSP; Boster). Antibody against β‐actin (Sigma‐Aldrich) was used as normalizing control. The membranes were subsequently incubated with the secondary antibodies for 1 hour at room temperature. The results were analysed using an Odyssey 2‐colour infrared laser imaging system (LI‐COR Biosciences, Lincoln, NE, USA). Relative density of labelled protein band was analysed by Image‐ProPlus 5.0 software (Media Cybernetics Inc, Rockville, MD, USA)
Odyssey 2 colour infrared laser imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey 2-colour infrared laser imaging system is a lab equipment product offered by LI-COR. It is a multi-channel fluorescence imaging system that utilizes infrared laser technology to detect and quantify proteins and other biomolecules in various applications, such as Western blotting, cell-based assays, and tissue imaging.
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Lab products found in correlation
Image pro plus 5, by Media Cybernetics (2 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Phospho gsk3β, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Phospho akt p akt, by Cell Signaling Technology (1 mentions)
T gsk3β, by Cell Signaling Technology (1 mentions)
Runx2, by Boster Bio (1 mentions)
β actin, by Merck Group (1 mentions)
2 protocols using odyssey 2 colour infrared laser imaging system
1
Western Blot Analysis of Osteogenic Markers
2
Western Blot Protein Analysis Protocol
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The cells were lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS with complete protease inhibitor cocktail). The protein concentration was evaluated using the BCA protein assay kit (Pierce). Protein (40 μg) from each cell extract was separated with electrophoresis on sodium dodecyl sulphate polyacrylamide gels and was transferred to a nitrocellulose membrane. Next, the membranes were blocked for 1 hour at room temperature with 5% nonfat milk and were incubated at 4°C overnight with diluted primary antibodies against Bmi-1 (Cell Signaling Technology) and β-actin (Sigma-Aldrich, St. Louis, MO, USA). The membranes were finally incubated with secondary antibodies for 1 hour at room temperature and were analysed using an Odyssey 2-colour infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA). The relative intensity of labelled protein bands was quantified using Image-Pro Plus 5.0 software (Media Cybernetics Inc., Rockville, MD, USA).
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