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Azure BiosystemsWestern blotting in 6 steps. This is a complete guide to equipment, supplies, and reagents needed for successful Western blot analysis.
Keep reading for an in-depth overview of the Western blotting steps. We’ve also added links to the tools so you can quickly find exactly what you need 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 handcast 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 or polyvinylidene difluoride (PVDF). Transfer involves assembling a transfer “sandwich” in which the gel is placed next to the membrane and 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.
What you need:
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.
What you need:
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.
What you need:
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 or on film.
What you need:
Finally, the bound secondary antibodies are detected. For chemiluminescent detection, the blot is incubated with a chemiluminescent substrate and the emitted light detected using film or a digital imager. For fluorescent detection, the blot is imaged using a digital imager that has a light source to excite the fluorophore and has the correct filters to detect the emitted fluorescence.
What you need:
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).
What you need:
READ MORE: What is Total Protein Normalization?
EXPLORE: Azure Imaging Systems, Azure Sapphire Biomolecular Imager
Chemiluminescent blots can be stripped and re-probed for additional target(s).
