1.1.5 Bone Tissues

Operational Steps for Total Protein Extraction from Bone Tissue

This method is suitable for extracting total protein from various animal bone tissue samples, including samples of compact bone, cancellous bone, and cartilage tissue. Commercial bone tissue protein extraction kits can be purchased as an option—these kits can fully lyse osteocytes, osteoblasts, and osteoclasts, and the extracted protein can be used in downstream protein research such as Western Blot and protein electrophoresis.
Experimental Steps
1. Sample Collection
Take fresh bone tissue samples. After completely removing the periosteum, fully soak the samples in pre-cooled physiological saline (replace the saline once), then rinse with distilled water to remove blood and red blood cells.
2. Grinding
Crush the bone tissue, weigh it, and place it in a mortar filled with liquid nitrogen. Grind the tissue into a powder, ensuring that the liquid nitrogen does not completely evaporate. If the tissue is too dry and hard to grind with liquid nitrogen alone, a small amount of extraction buffer can be added directly before grinding.
3. Lysis
Transfer the bone tissue powder into a centrifuge tube. For every 100 mg of bone tissue, add 200 μL–300 μL of protein lysis buffer and an appropriate amount of protease inhibitor.
4. Mixing/Vortexing
After mixing, incubate the tube in an ice bath for 30 minutes. Vortex the mixture on a vortex mixer for 20 seconds every 5 minutes to prevent the powder from settling and ensure sufficient extraction.
5. Centrifugation
Centrifuge at 12,000 rpm at 4°C for 15 minutes.
6. Supernatant Collection
Transfer the supernatant to a new centrifuge tube—this supernatant is the total protein extracted from the bone tissue. The extracted protein can be aliquoted and stored at -80°C for later use.
7. Precautions:
(1) Do not aspirate the sediment at the bottom when transferring the supernatant.
(2) It is recommended to pre-cool all reagents used in the experiment in a -20°C refrigerator. Throughout the entire process, it is best to keep the samples at a low temperature.
(3) If no protease inhibitor is added in the commercial extraction kit, 1× working concentration of protease inhibitor can be added in Step 3.