Polyclonals are usually produced in rabbits, donkeys, sheep, and goats, and are purified from serum. Polyclonal antibodies consist of a mixed pool of immunoglobulin molecules that bind to several different epitopes found on a single antigen. The antibodies used to detect the target protein in a western blot will be either monoclonal or polyclonal. You might also want to consider treating cells with growth factors or chemical compounds that induce or inhibit expression of the target. When selecting a phospho-specific antibody for your experiments, it is crucial to ensure that the antibody specifically detects the protein of interest only when it is phosphorylated at the indicated site.

The wording good and bad antibody or the most specific antibody should be avoided, since a specific antibody in one sample context can give rise to high cross-reactivity in another sample context depending on the nature of the epitope(s) that it will recognize. Before performing the experiments review the literature in order to understand the expression profile of the protein

As a negative control, you can include secondary-antibody-only controls (omitting the primary antibody incubation step) and samples from a tissue or cell lysates known not to express the target protein such as cells engineered with a genetic knockout. As a positive control, purified proteins or lysates overexpressing the target protein can be used. To ensure the specificity of the primary antibody, it is important to use positive and negative controls when running your blot. Primary Antibody SelectionĪntibodies should be specific, selective, and give reproducible results. Antibodies can be designed to be specific for certain parts of a target protein, or they can be designed to be specific for only modified versions of the protein. Primary antibodies are raised against a protein of interest and will selectively recognize and bind to target proteins that have been immobilized to a membrane. Avoid use of nonfat dry milk and instead use BSA, casein, or advanced formulations that are compatible with phosphoprotein detection. Advanced formulations like Bio-Rad’s EveryBlot Blocking Buffer can complete the blocking step in 5 minutes.įor detection of a phosphorylated form of a protein, blocking buffers should not contain phosphorylated proteins to avoid high background signal. In contrast, insufficient incubation times will increase nonspecific binding of the primary antibody. Excessive blocking times may result in lower sensitivity as epitopes can be masked by the blocking agent and proteins washed off the membrane. Blocking for 1 hour with constant agitation is a good starting point. Modern formulations like Bio-Rad’s EveryBlot Blocking Buffer are universal, performing well across a wide range of targets, sample types, and detection methods.īlocking duration also affects the signal and background levels. Optimize the detection system for maximal signal with lowest background by testing several blocking agents. The best blocker for each experiment will depend on the antibody and membrane type. Various blocking reagents are available, and no single blocker will be optimal for all antibody/antigen combinations. Failure to completely block these sites can lead to high backgrounds that obscure the signal. Some experiments require only a qualitative answer, but properly designed and executed western blot experiments can also provide quantitative data on relative protein expression between samples.Following transfer, unoccupied antibody binding sites on the membranes must be blocked to prevent nonspecific binding. Understanding basic imaging concepts such as sensitivity, resolution, and sources of background noise can help you maximize image data quality.Īn image of a western blot is rich with information. In addition to detection of specific proteins, the total protein in a sample can also be visualized with total protein stains or newer technologies that eliminate the need for staining and destaining.Īccurate imaging of your western blot is crucial for capturing blot data for downstream analysis. Efficient protein transfer is required for maximum western blot sensitivity.Ī good, clean western relies on the specificity and sensitivity of your antibodies. Separated proteins are transferred from the gel to a membrane where they are immobilized. Good sample preparation techniques ensure proteins remain undamaged for downstream analysis.Įlectrophoresis separates the proteins in the sample and provides molecular weight data for detected proteins during subsequent detection. Cells containing your protein of interest must be lysed completely to ensure a high yield while removing non-protein components of cells.

Bio rad western blot troubleshooting