The right antibodies are essential for clean, definitive, and reproducible western blot results. We offer more than 40,000 highly specific and sensitive primary and secondary antibodies to help you gather quality western blot data. All of our antibodies are validated to perform in the stated application and species. HRP- and AP-conjugated secondary antibodies are also available in various degrees of purity to meet all your western analysis needs.
- Specific antibodies to bind your target protein or primary antibody
- Sensitive antibodies to give you the level of detection you need
- Reliable antibodies to help you get great data every time
- Validated antibodies to perform in the stated application and species
- Affordable antibodies to help you get the most out of your research budget
Primary antibodies for western blot
Antibodies are critical to the success of the western blot technique. They allow for the selective detection of the protein of interest. Typically, a primary antibody is used to specifically bind the protein of interest and a labeled secondary antibody is used for detection.
Both polyclonal and monoclonal antibodies work well for western blotting. Polyclonal antibodies are less expensive and less time-consuming to produce, and they often have a high affinity for the antigen. Monoclonal antibodies are valued for their specificity, purity and consistency that result in lower background. Both polyclonal and monoclonal antibodies are developed using mouse, rat, rabbit, goat, and other animal species as hosts. Crude antibody preparations such as serum or ascites fluid are sometimes used for western blotting, but the impurities present may increase background.
Primary antibodies are typically diluted from their stock concentration prior to use, and each antibody requires some optimization in order to perform at its best (always check your product data sheet for recommended dilutions). After the blot is incubated with the primary antibody solution, the blot is washed, and if the primary antibody is not labeled with a detection molecule, a secondary antibody or other secondary detection reagent is added.
Primary antibodies can also be used to detect loading controls during western blot. Loading control antibodies are helpful for assessing western blot efficiency and to compare the amount of protein loaded in each well across the gel. These controls help determine whether sample-to-sample expression level differences are due to actual protein levels or from loading variance.
Top loading control antibodies
Secondary antibodies for western blot
Secondary antibodies can be conjugated to a number of different molecules for detection, such as enzymes, fluorophores, and dyes. The most common secondary conjugates are horseradish peroxidase (HRP) or alkaline phosphatase (AP) enzyme. Fluorescently labeled secondary antibodies, like our selection of Alexa Fluor antibodies, are also becoming popular. Fluorescence-based detection provides sensitivity similar to that of chemiluminescence detection but allows for the detection of multiple fluorophores at the same time to give comparative data for two or more different proteins.
The use of secondary antibodies can greatly increase sensitivity compared to the use of a labeled primary antibody. Directly conjugated primary antibodies usually have a small number of labels conjugated per antibody. Secondary antibodies are designed to bind the primary antibody in more than one place, which results in several secondary antibodies being bound to the primary antibody. This results in a 3- to 5-fold increase in the number of labels or enzyme present and a significant amplification in signal.
Secondary antibodies are typically diluted from their stock concentration prior to use, and each antibody requires some optimization in order to perform at its best (always check your product data sheet for recommended dilutions). After the blot is incubated with the secondary antibody solution, the blot is washed, and treated with a substrate to visualize the bands.
Top secondary antibodies for western blotting
Example western blot data
Figure 1. Rabbit igG (H+L) Secondary Antibody in WB. Endogenous MEK2 (left) was detected using rabbit anti-MEK2 ABfinity monoclonal primary antibody and goat anti-rabbit IgG (H+L) Superclonal secondary antibody, Alexa Fluor 790 conjugate. The control blot (right) was not treated with primary antibody to demonstrate lack of cross-reactivity of the secondary antibody. Western blot analysis was performed on whole cell extracts of HeLa, human cervical carcinoma, cells (lane 1–5) using the XCell Surelock Electrophoresis System and iBlot Dry Blotting System.
Figure 2. α-Tubulin Antibody in WB. α-Tubulin was detected using α-Tubulin mouse monoclonal primary antibody and goat anti-mouse HRP conjugate secondary antibody. Western blot analysis was performed on cell lysates from 8 cell lines (see above) using the XCell Surelock Electrophoresis System and iBlot Dry Blotting System.
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