Western Blot (WB), also referred to as an immunoblot, is an analytical technique that uses antibodies to detect specific proteins within a sample. It typically follows an SDS-PAGE (see protocol) to further characterise proteins that had previously been separated on relative molecular mass only. A target protein-specific primary antibody (in solution) is washed over the gel electrophoresis membrane. This is followed by 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.
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
- Blotting Buffer
- 48 mM Tris base
- 39 mM Glycine
- 20% Methanol
- pH ~9.2
- Wash Buffer: 0.05% Polysorbate 20 in PBS
- Blocking Buffer: 5% BSA, 0.05% Polysorbate 20 in PBS
- Cling Film
- Primary Antibody
- Secondary Antibody
Transfer to Membrane (WB)
- Incubate gels in blotting buffer for 10 mins.
- Activate PVDF membrane in methanol for 1 min before equilibrating in blotting buffer for 5 mins. Note: Nitrocellulose membrane does not need activating.
- 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.
- Incubate membrane using Ponceau S solution for 10 mins to visualise and confirm protein transfer.
- Remove excess stain by rinsing membrane in 5% acetic acid solution.
- 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.
- Remove Ponceau S stain using 0.1 M sodium hydroxide solution. Wash with water.
- 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.)
- Wash the membrane with wash buffer for 5 mins on a shaker at room temperature. Repeat twice.
- Incubate the membrane with primary antibody at appropriate concentration in blocking buffer for ~1-2 hrs on shaker at room temperature.
- Wash (repeat step 2).
- Incubate the membrane with secondary antibody at appropriate concentration in blocking buffer and incubate for ~1 hr on shaker at room temperature.
- Wash (repeat step 2).
- Transfer membrane onto cling film, protein side up.
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.