Extract proteins

Extract proteins into 50 mM NH4HCO3 (pH 8.0) with 1 % sodium deoxycholate (ABC/DOC).

         Option 1: For cultured or primary cells:

  1. Wash cells first in PBS.
  2. Pellet at 600 x g.
  3. Remove supernatant.
  4. 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:

  1. 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.
  2. Pulverize the tissue in a beadmill.
    Determine the specific conditions empirically for each tissue and beadmill type.
  3. After milling, pellet insoluble material at 16,000 x g for 10 min at 4˚C.
  4. Move the supernatant to a clean tube.
  5. 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).
  6. Allow the solution to stand at RT for 90 min.
  7. Pellet the proteins by centrifuging for 10 min at 16,000 x g.
  8. Resuspend the pellet from this final step in ABC/DOC to bring the protein concentration to 1 µg/µl.