The increased sensitivity of conjugated secondary antibodies, compared to primary antibody only, results from these antibodies binding to the primary antibody at multiple locations, which amplifies the signal. Remember to check that the light emission wavelength of a conjugate is compatible with your reading device/scanner. Using an enzyme-linked secondary antibody, such as horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-conjugate antibody, or a western blot-optimized fluorescence conjugate, offers a high level of sensitivity. To visualize your protein, select a secondary antibody (against the host species of the primary antibody) that will bind to the primary antibody. Whenever possible, choose a primary antibody that has been knockout (KO) validated to ensure it binds specifically to the intended target. The choice of host species of the primary antibody is less critical when using samples that don’t contain endogenous immunoglobulin. This is to avoid cross-reactivity of the secondary anti-immunoglobulin antibody with endogenous immunoglobulins in the sample. For example, if you are studying a mouse protein, choose a primary antibody raised in a species other than mouse (eg, primary antibody raised in rabbit). When working with tissue lysates or tissue culture supernatants containing serum and, therefore, endogenous immunoglobulins, you should select a primary antibody raised in species different from that of your sample. The sensitivity and specificity of your western blot depend on the quality of the antibodies and the experimental conditions they are used in. The membrane can be further processed with antibodies specific for the target of interest and visualized using enzyme-linked or fluorophore-conjugated secondary antibodies and detection reagents. Protein transfer to the membrane is essential because gels used for electrophoresis provide a poor surface for immunostaining, ie, antibodies don’t stick to the proteins in the gel. The gel is then placed in contact with the membrane, and the use of an electrical current induces the proteins to migrate from the gel to the membrane. In the first step, the proteins are separated based on size by gel electrophoresis. To achieve this, western blot implements three steps: (1) separation by size, (2) transfer to a solid support, and (3) visualizing target protein using primary and secondary antibodies. Western blot allows us to determine the relative protein levels between samples and establish the molecular weight of the target, which can provide insight into its post-translational processing. Western blot, or western blotting, is a technique widely used in research to separate and identify specific proteins within a complex mixture.
0 kommentar(er)
