Western blot protocol

Western blot protocol

20161011 Protocole WB EN
Needed products:

Proteins preparation and separation by gel electrophoresis

  1. Remove a small volume of protein sample to determine protein concentration
  2. Take 20 µg of proteins and add Laemmli
  3. Boil 5 minutes at 95°C, then spin the tubes
  4. Load equal volume of proteins into the wells of the SDS-PAGE gel (acrylamide%depend on the size of the target protein), not forget a well for control weight ladders.
  5. Migration at constant amperage with appropriate buffer

Protein transfert from the gel to the membrane

  1. Place the sandwich (gel/membrane/blotting paper) in the transfer box and make sure no air bubbles are trapped
  2. Transfer at constant voltage with the appropriate buffer

Antibody incubation and revelation

  1. Stain the membrane with Ponceau red to check the protein transfert quality
  2. Rince off the membrane and incubate in blocking solution
  3. Incubate overnight with the primary antibody at 4°C
  4. Rince the blot
  5. Incubate with the secondary antibody 1h at room temperature
  6. Rince the membrane
  7. Apply the substrate to the blot