Western Blot

Western Blot (WB), is a widely used analytical technique that uses antibodies to separate and identify specific proteins in a complex biological sample. It typically follows an SDS-PAGE (see protocol). 

Gel electrophoresis is used to separate sample proteins based on their relative molecular mass. Proteins are then blotted to a membrane and incubated with a primary antibody specific to the target protein. This is followed by incubation with a conjugated-secondary antibody, which allows the visualisation of the target protein through methods such as staining, immunofluorescence and radioactivity.

See below for a general WB protocol.

A. Reagents

for Transfer to Membrane (WB)

  • PVDF membrane (0.4 µm) or nitrocellulose membrane (0.45 µm)
  • Transfer equipment
  • Transfer pads and filter paper
  • 2X Protein Loading Buffer
  • Ponceau S Solution
  • 5% Acetic acid
  • 0.1M NaOH

for Immunoblotting

  • Wash Buffer: 0.05% Polysorbate 20 in PBS
  • Blocking Buffer: 5% BSA, 0.05% Polysorbate 20 in PBS
  • Cling Film
  • Primary Antibody
  • Secondary Antibody

B. Protocol

Transfer to Membrane (WB)

  1. Incubate gels in blotting buffer for 10 mins.
  2. Activate PVDF membrane in methanol for 1 min before equilibrating in blotting buffer for 5 mins. Note: 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 1hr. Note: Transfer times depend on protein size.
  4. Incubate membrane using Ponceau S solution for 10 mins 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. Note: 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.

Immunoblotting

  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 mins on a shaker at room temperature. Repeat twice.
  3. Incubate the membrane with primary antibody at appropriate concentration in blocking buffer for ~1-2 hrs 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 hr on shaker at room temperature.
  6. Wash (repeat step 2).
  7. Transfer membrane onto cling film, protein side up.

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.

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