The six Western blot steps outlined below serve as the complete guide to everything you need for successful Western blot analysis, including equipment, supplies, and reagents.
Keep reading for an in-depth overview of the six Western blotting steps. We’ve also added links to the tools you’ll need, so you can quickly find exactly what’s required to complete your experiments.
For most experiments, the first step to Western blot analysis is separating the proteins in a sample using polyacrylamide gel electrophoresis (PAGE). In SDS-PAGE, the proteins in the sample are coated with the detergent SDS. The proteins then migrate according to their size, with smaller proteins migrating more quickly through the gel. The percentage of polyacrylamide in the gel determines how easily proteins of various sizes can move through the gel. Higher percentage gels having a tighter gel matrix better for resolving smaller proteins.
Polyacrylamide gels may be purchased ready-to-use in a variety of percentages or gradients. You can also choose to hand cast your own gels to achieve a customized percentage.
Once protein separation is complete, the proteins are transferred from the polyacrylamide gel to a solid membrane support. Membranes are usually made from nitrocellulose (NC) or polyvinylidene difluoride (PVDF). The transfer step involves assembling a transfer “sandwich” (Figure 1) in which the gel is placed next to the membrane. Both are placed inside a cassette with blotting paper and sponges on either side to ensure a secure fit within the cartridge. Transfer occurs after the sandwich submerged in transfer buffer in a tank, and a current is passed through the sandwich to drive the proteins from the gel to the membrane.
Before the target protein(s) can be detected on the Western blot, non-specific binding sites on the membrane must be blocked. This is done by incubating the membrane in a blocking buffer. For ease of use, premade blocking buffers are available for purchase on the market. More details are found below. Homemade blocking buffers contain proteins, such as dry milk or serum albumin, to block non-specific protein-binding sites.
The blocked membrane is incubated with an antibody that binds to the target protein of interest. Incubation conditions depend on the antigen-antibody pair. The primary antibody may be diluted in blocking buffer. Learn more about primary antibodies at Bosterbio. Excess unbound primary antibody is washed away in a series of washes.
The presence of primary antibodies bound to their target protein on the blot is detected by binding a labeled secondary antibody to the primary antibody. Secondary antibodies are usually labeled with a fluorophore that can be detected directly, or bound to an enzyme like horseradish peroxidase (HRP). HRP can react with a substrate to produce light (chemiluminescence), or a colored product that can be detected visually using an Azure Imager, another digital imager, or using film.
Finally, the bound secondary antibodies are detected. For chemiluminescent detection, the blot is incubated with a chemiluminescent substrate and the emitted light detected using a digital imager like the Azure 300 Imager, or using film in a dark room.
For fluorescent detection, the blot is imaged using an imager such as the Azure 500 that has a light source to excite the fluorophore and the correct filters to detect the emitted fluorescence.
Digital blot images can be analyzed using analysis software, such as AzureSpot Pro. To obtain quantitative information from a Western blot, the signal for the protein of interest can be compared to the signal for a housekeeping protein or to total protein in a process known as total protein normalization (TPN).
Need help with your Western blots and don’t know where to begin?
FREE WESTERN BLOT eBOOK
Related blog posts on Western blotting…
Validating your antibodies is the process of confirming an antibody recognizes your protein of interest with low cross-reactivity to other targets. Validating antibodies is critical