As a biochemical technique providing information to characterize proteins in complicated samples, Western blotting, or immunoblotting, is used in several scientific and medical sectors. It’s popular due to its simple process and cost-effective nature, making it common in studies and research. Despite this common use, it can be frustrating when challenges start. Issues can occur at any stage during the process, no matter how experienced the researcher. During these five stages, the technique can fail at any point. However, with new technologies emerging, they can be used to identify failed western blogs and streamline the process.
The 5 Stages of Western Blotting
To monitor changes in proteins, such as modifications and expressions, western blots recognize the protein’s quantity, molecular weight and post-translational changes. Here’s an overview of the five stages of the process:
- Sample Preparation. Buffers are used to lyse sample cells, which makes the targeted proteins available in suspension. The proteins can be unfolded and given a negative charge, with researchers breaking the sample down through homogenization, sonication or blending in some situations.
- Protein Separation. Electrophoresis is used to separate proteins based on their mass charge ratio. The sample is forced through a gel, separating the charged molecules based on the physical properties. This allows researchers to separate proteins by molecular weight, isoelectric point or electric charge. The most common form, SDS-PAGE, uses a polyacrylamide gel with sodium dodecyl sulfate buffers to separate proteins by mass.
- Blotting. The separated proteins are transferred from the gel onto blotting membranes. The membrane is typically polyvinylidene diflouride (PVDF) or a nitrocellulose base. This makes the proteins accessible to antibody detection. Transfer can be done by diffusion, vacuum blotting, capillary and electroblotting, the last of which is faster adan more efficient than other methods, using an electric current to pull proteins from gel to membrane.
- Antibody Incubation. Researchers prevent any non-specific antibody binding by using buffers such as milk, serum or highly-purified proteins to block the membrane. A primary antibody is bound to the target protein, with excess washed off. A tagged secondary antibody is then introduced to stick to the primary antibody, which detects target antigens, providing a readout for imaging purposes.
- Imaging/Data Analysis. The secondary antibody reporter molecule is detected using a film, scanner or CCD camera. This allows the researchers to analyze the sample and establish the target protein’s quantity, size and presence.
New Technology for the Western Blotting Technique
With the advent of digital transformation, new technologies have emerged to tell researchers when issues arise during the process. Some are taking a stain-free approach, which can ensure that the blot has been completed successfully before detection agents and antibodies are applied to failed blogs. This has the potential to save significant time, money and effort for the scientific community.
As a versatile technique that provides strong benefits in many applications, the Western blog can be used for anything from simple research to advanced diagnoses determination. Often very useful early on during studies, it’s commonly used to validate a successful transfection of cell colonies using non-viral vectors. It can also be used to identify specific proteins within a sample, while rudimentary information about the protein’s size can be determined at the same time. Given the western blot’s ability to conform makes it a technique to use later in the study, especially when confirming how successful gene editing techniques such as CRISPR have been.