Western blotting is a technique that uses specific antibodies to identify proteins separated by size through gel electrophoresis.
The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane, and an electrical current is applied. This induces the proteins to migrate from the gel to the membrane. The membrane can be further processed with antibodies specific to the target of interest and visualized using secondary antibodies and other detection reagents.
Before running a western blot, we must make our protein of interest accessible to the antibodies. This usually involves preparing a lysate to extract the protein from cells or tissues. Lysates can be diluted into several aliquots in a loading buffer and stored frozen at -80 °C until ready for use.
Prepare a lysis buffer according to the manufacturer’s instructions.
Keep samples, buffers and equipment on ice throughout the process.
Isolate your cells and suspend them in lysis buffer.
Practice aseptic technique while handling cells.
Lyse the cell suspension.
Time and intensity for sonication will vary between different instruments, so some optimization is required.
Spin down the suspension to pellet insoluble contents.
Keep the supernatant in place in a fresh tube on ice.
This supernatant is now your lysate.
Determine the protein concentration of your lysate using a Bradford or BCA assay.
If the protein concentration at this stage is low, and your protein resides in the nucleus or mitochondria, you could consider fractionating your original sample to produce a more concentrated lysate.
We offer cell fractionation kits for this purpose.
Aliquot the lysate into several tubes.
Leave enough space to add adequate loading buffer.
Dilute the aliquots in loading buffer.
Diluting lysates in loading buffer prior to storage in the freezer makes them more stable.
Store samples at -80°C until ready for use.
The gel is immersed in buffer, the protein samples are loaded, and an electrical current is applied to the gel, which causes proteins to migrate from one end of the gel (negative electrode) to the other (positive electrode). Proteins are separated by size; smaller proteins travel more quickly through the gel, so appear further down.
To confirm the size of each protein in your sample, they are run alongside molecular weight ladders.
SDS-PAGE gel (Tris-Glycine, Bis-Tris or Tris Acetate based gel)
Molecular weight ladder (example ab116028)
Gel running apparatus
SDS
Running buffer (example Tris-Glycine, MES, MOPS, Tris-Acetate)
Select an appropriate SDS-PAGE gel for your protein and set up the running apparatus.
Table 1: Recommended gradient gel chemistries for different protein sizes. Our lab use gradient gels, but gels with fixed acrylamide concentration can also be used.
| Protein size | Recommended gel and buffer system |
|---|---|
| 10–30 kDa | 4–12% acrylamide gradient Bis-Tris gel MES running buffer |
| 31–150 kDa | 4–12% acrylamide gradient Bis-Tris gel MOPS running buffer |
| > 150 kDa | 3–8% acrylamide gradient Tis Acetate gel Tris Acetate running buffer |
Table 2: Recommended gel chemistries to use for fixed-concentration Tris-Glycine gels. Some optimization will be required if preparing your own gels; a 10 - 15 % separating gel is often a good starting point.
| Small proteins | Average proteins | Large proteins |
|---|---|---|
| > 4 kDa | 12–100 kDa | < 200 kDa |
| 20% separating gel | 10–15% separating gel | 8% separating gel |
| Tris-Glycine running buffer | Tris-Glycine running buffer | Tris-Glycine running buffer |
Larger proteins should have a lower percentage of acrylamide in the gel. This creates a less dense polymer that is easier for proteins to migrate through.
When setting up the running apparatus, make sure the positive and negative electrode are plugged in the right way round.
Thaw and fully denature your lysates.
Load an equal mass of protein from each sample into the gel.
Take care not to touch the bottom of the wells with the pipette tip, as this can create a distorted band.
Make sure the wells are straight before adding samples.
After loading, the denatured lysates prepared in Step 4 can be stored at -20 °C for future use.
Run the gel according to the manufacturer's instructions.
Ideal running times and voltages can vary according to the manufacturer, the gel composition, and the protein of interest. Larger proteins should be run at a higher voltage for a longer time.
Remove the gel from the running apparatus when ready to transfer.
After performing electrophoresis, proteins are then transferred (or ‘blotted’) onto a membrane ready for antibody incubation. This membrane can be made of nitrocellulose or polyvinylidene fluoride (PVDF); either material is acceptable.
As with electrophoresis, transfer to the membrane is achieved by applying an electrical charge, which causes the proteins to migrate. The proteins travel away from the gel near the negative electrode and towards the positive electrode, where they bind to the membrane.
Semi-dry transfer requires additional equipment but has a much shorter transfer time with easier setup.
Your SDS-PAGE gel
Transfer apparatus
Wash buffer (example TBST)
Membrane (either nitrocellulose or PVDF)
Methanol (if using PVDF)
Soak the membrane in methanol, if using a PVDF membrane.
This step is essential for PVDF membranes but not needed for nitrocellulose membranes. As PVDF is naturally hydrophobic, it requires activation with methanol to allow the buffer to pass through effectively.
Soak the membrane in water, then in transfer buffer for 10 min at 4 °C.
Assemble the SDS-PAGE gel and the membrane in the transfer cassette.
Make sure the filter paper is cut to the same size as the membrane.
For detailed guidance on assembling the transfer apparatus, refer to the manufacturer’s instructions.
Run the transfer according to the manufacturer's instructions.
Ideal running times and voltages can vary according to the manufacturer. Optimization may be required.
Remove the gel and membrane from the transfer apparatus.
Before proceeding, you can check the protein has successfully transferred to the membrane.
You can check the success of the transfer using Coomassie staining of the gel.
You can use the pre-stained molecular weight ladder as an initial check to compare the amount of protein on the PAGE gel and the membrane.
For the best quality results, Ponceau S staining is not recommended for fluorescent western blot because it can lead to high background fluorescence, even after extensive washing. There are alternative protein stains that do not fluoresce.
Your membrane
Your SDS-PAGE gel
Coomassie stain (example ab119211)
Observe the colored bands of the pre-stained molecular weight ladder.
Stain the SDS-PAGE gel in Coomassie stain.
Even if your transfer is successful, there will still be some proteins on your SDS-PAGE gel after transfer. You’re aiming for the gel to be mostly clear.
Use the procedures below for antibody incubations. If using loading control antibodies in chemiluminescent western blot, the staining procedure below can be repeated on the same membrane after stripping.
In fluorescent western blot, the membrane can be incubated with multiple sets of antibodies simultaneously according to the following procedure. Loading control antibodies and detection antibodies can be run on the same membrane without the need for stripping.
Blocking buffer (example: 3–5% milk or BSA in TBST, or non-mammalian protein buffer)
Wash buffer (example TBST)
Your membrane
Conjugated primary antibody
Place the membrane in a container and cover with blocking buffer.
The blocking buffer will contain milk or BSA (3–5% in TBST).
Generally, BSA will give clearer results as it contains fewer proteins for the antibody to cross-react with. Some antibodies will work better with milk as it contains a greater variety of blocking proteins.
When this is known, the blocking buffer will be advised on the antibody datasheet.
Dilute the antibody in blocking buffer to the recommended dilution.
Optimum dilutions will often be suggested on the antibody datasheet.
If not, you may need to perform serial dilutions to find the antibody concentration that works best.
Cover the membrane with primary antibody in blocking buffer.
Incubate with gentle rocking overnight at 4 °C, or 1 h at room temperature.
Incubation time may need optimization.
Wash the membrane three times with wash buffer, 5 min each.
Once incubation is complete, you’re now ready to image your western blot.
In chemiluminescent detection, the first step is to incubate the blot in a chemiluminescent substrate solution, which will cause light to be emitted where HRP-conjugated antibodies are present. These bands of light on the blot correspond to antibody binding and should be resolvable as bands on the blot. Chemiluminescent blots have been traditionally imaged using X-ray film-based techniques, but these are largely being replaced by benchtop charge-coupled device (CCD) imagers, which provide much higher quality images.
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.
Clean the imaging scanning bed with 70% ethanol using a cotton lint-free cloth.
Keep the scanning bed as clean as possible to avoid background fluorescence.
Scan membranes in the imaging system.
Scanning membranes wet works well with histones.
Remove the membranes from the scan bed and clean the imaging scan bed with 70% ethanol using a cotton lint-free cloth.
Keep the scanning bed as clean as possible to avoid background fluorescence.
Stripping the membrane allows you to remove antibodies from the membrane and restain it with a different set of antibodies. This is helpful if you intend to incubate the membrane with loading control antibodies.
Strip the membrane with stripping buffer.
Use a volume of buffer that will completely cover the membrane.
Wash the membrane in PBS for 5 min at room temperature.
Incubate the membrane with a small amount of ECL detection reagent.
Proteins can be identified by bands at or near the expected molecular weight, as confirmed by the molecular weight ladder. To rule out non-specific interactions, the same band should be absent in the negative control lane. For example, in the figure above, we see that the negative control lane does not have a band for the target (CD133).
Note that bands can differ from the expected molecular weight for a range of reasons, including:
If the bands are at an unexpected molecular weight or difficult to resolve in any other way, please refer to our troubleshooting guide.