Western Blot Antibodies

Western blotting is the process of separating a heterogeneous mixture of proteins by polyacrylamide gel electrophoresis (PAGE) before transferring them to a membrane for antibody-based detection. It can broadly be divided into native western blotting, which separates proteins based on both size and charge and is useful for studying protein complexes or enzymatic activity, and denaturing and reducing western blotting, which is more common. Denaturing and reducing western blotting uses the detergent sodium dodecyl sulfate (SDS) and a reducing agent such as β-mercaptoethanol to revert intact proteins back to their primary structure and give them a strong negative charge, meaning they are separated by size alone. A typical western blotting workflow involves several main steps. First, the samples (typically cell lysates or tissue homogenates) are prepared in an appropriate loading buffer such that they all share the same concentration. The samples are then loaded onto the gel alongside a molecular weight marker and relevant biological controls. Next, an electrical current is applied to the gel, causing the proteins to migrate towards the anode. Following separation, the proteins are transferred to a nitrocellulose or polyvinyl difluoride (PVDF) membrane using a specialized transfer apparatus. The membrane is then blocked to prevent non-specific antibody binding and incubated with a primary antibody raised against the target of interest. After this, a series of washes is performed and a labeled secondary antibody is added to enable target detection. Finally, after further washing to remove any unbound reagents, the blot is imaged and the bands may be quantified using a suitable software package. When selecting western blot antibodies, researchers must choose between direct or indirect detection. Direct detection uses labeled primary antibodies, thus shortening the western blotting workflow and allowing for multiplexing with antibodies sharing the same host species. Indirect detection instead uses a combination of unlabeled primary antibodies and labeled secondary antibodies, which can provide signal amplification. Another consideration is whether to use monoclonal or polyclonal antibodies. Monoclonal antibodies provide the advantages of highly reproducible performance and guaranteed long-term supply, while polyclonal antibodies may increase the likelihood of detecting more challenging targets due to exhibiting epitope redundancy. The type of antibody label is also important. Enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are used for enhanced chemiluminescent (ECL) detection, which provides high sensitivity but produces a signal with only a finite lifetime. Fluorophore labels like APC, FITC, PE, Cyanine, and PerCP dyes enable multiplexing and have a long-lived signal provided the blots are stored in the dark. Other factors to consider include the type of loading control, which could be a housekeeping protein (e.g., actin, tubulin, or GAPDH) or involve total protein normalization, and whether an epitope tag antibody could be used instead of a target-specific primary antibody. Epitope tag antibodies recognize moieties such as His, Myc, or V5, which are often added to the termini of recombinant proteins to enable their isolation or detection. Whichever method is chosen, optimization is essential to ensure accurate results.