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 (where the proteins in the sample are coated with the detergent SDS), proteins 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, with 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, or gels may be hand cast 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). Transfer involves assembling a transfer “sandwich” (Figure 1) in which the gel is placed next to the membrane. Both are then placed inside a cassette with blotting paper and sponges on either side to ensure a secure fit within the cartridge. Transfer occurs with 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 blot, non-specific binding sites on the membrane must be blocked by incubating the membrane in a blocking buffer. Home-made 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) that reacts 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 an Azure 300 Imager, another digital imager, or in a dark room using film. 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.
The Azure 500 is a multichannel, multimodal fluorescent imager, with IR, visible light, and UV excitation channels. It allows you to image and quantify two different targets in the same position more efficiently using Near-Infrared (NIR), and normalize to fluorescent total protein stain or a housekeeping protein in the green channel without needing to strip and re-probe your Western blots.
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).
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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