184.108.40.206. Extract proteins
Option 1: For cultured or primary cells:
- Wash cells first in PBS.
- Pellet at 600 x g.
- Remove supernatant.
- Solubilize the pellet in ABC/DOC at a ratio of 100 µl per 2E+07 cells.
The final protein concentration, measured by the BCA method, should be approximately 1 µg/µl.
Option 2: For any bee tissues:
- Place the material in a beadmill tube with enough 6 M urea, 2 M thiourea and 50 mM Tris (pH 8.0) to fully immerse the tissue.
- Pulverize the tissue in a beadmill.
Determine the specific conditions empirically for each tissue and beadmill type.
- After milling, pellet insoluble material at 16,000 x g for 10 min at 4˚C.
- Move the supernatant to a clean tube.
- Precipitate the proteins in the supernatant by adding four volumes of 100% ethanol, 20 µg of molecular biology-grade glycogen and 50 mM sodium acetate (pH 5, from a 2.5 M stock solution).
- Allow the solution to stand at RT for 90 min.
- Pellet the proteins by centrifuging for 10 min at 16,000 x g.
- Resuspend the pellet from this final step in ABC/DOC to bring the protein concentration to 1 µg/µl.