Western blot

C. Transfer to membrane (Western Blotting)

Reagents

  • PVDF membrane (0.4 ?m) or nitrocellulose membrane (0.45 ?m)
  • Transfer equipment
  • Transfer pads and filter paper
  • 2X Protein Loading Buffer
  • Ponceau S Solution
  • 5% Acetic acid
  • 0.1M NaOH
  • Blotting Buffer
    • 48 mM Tris base
    • 39 mM Glycine
    • 20% Methanol
    • pH ~9.2
  1. Incubate gels in blotting buffer for 10 min.
  2. Activate PVDF membrane in methanol for 1 min before equilibrating in blotting buffer for 5 min. (NB. Nitrocellulose membrane does not need activating.)
  3. Assemble the gel, membrane, filter paper and pads in the blot transfer cell (pad/paper/gel/membrane/paper/pad). Transfer at 100V for 1h. (NB. Transfer times depend on protein size.)
  4. Incubate membrane using Ponceau S solution for 10 min to visualise and confirm protein transfer.
  5. Remove excess stain by rinsing membrane in 5% acetic acid solution.
  6. Cut the membrane for staining with different antibodies. (NB. Use a pencil to label tracks on the membrane and indicate which side the protein is on.)
  7. Remove Ponceau S stain using 0.1 M sodium hydroxide solution. Wash with water.

D. Immunoblotting

Reagents

  • Wash Buffer
    • 0.05% Polysorbate 20 in PBS
  • Blocking Buffer
    • 5% BSA
    • 0.05% Polysorbate 20 in PBS
  • Cling Film
  • Primary Antibody
  • Secondary Antibody
  1. Incubate the membrane in blocking buffer at 4°C overnight. (NB. Membrane can be dried and stored at 4°C. PVDF membrane must be reactivated by soaking in methanol for 1 min.)
  2. Wash the membrane with wash buffer for 5 min on a shaker at room temperature. Repeat twice.
  3. Incubate the membrane with primary antibody at appropriate concentration in blocking buffer for ~1-2 h on shaker at room temperature.
  4. Wash (repeat step 2).
  5. Incubate the membrane with secondary antibody at appropriate concentration in blocking buffer and incubate for ~1 h on shaker at room temperature.
  6. Wash (repeat step 2).
  7. Transfer membrane onto cling film, protein side up.

E. Detection

Detection will depend on the conjugation of your primary or secondary antibody. For fluorescently labelled antibodies, use a fluorescence scanner, as per the manufacturer’s instructions. For HRP conjugated antibodies, use an enhanced chemiluminescence (ECL) kit and follow manufacturer’s guidelines.


To SDS-PAGE