In any kind of immunostaining experiment, your research can only be as good as the antibody you apply. Inappropriate antibody usage can lead to inaccurate results that are not consistent or reproducible. In order to obtain a specific and robust signal, you should use a high-quality antibody with minimal cross-reactivity that has been validated for your application and has been optimized for your sample. The experts at R&D Systems have provided us with tips on how to find just such an antibody.
With so many vendors available, it is important to understand what to look for from a supplier. Here is a list of questions to consider when deciding where to buy your antibody.
Was the antibody manufactured by the supplier or was it purchased and resold? If the supplier manufactures its own products, it has complete control over production, quality control, and shipping and storage conditions—basically, the experts handle everything. In addition, when the technical support group is associated with the scientists who actually make the products you will likely receive better support when it’s needed.
Has the antibody been quality controlled? The process of antibody development should begin with immunogen optimization and proceed with extensive validation. At R&D Systems, antibodies are manufactured under controlled conditions and then undergo rigorous tests to ensure consistency, reproducibility, and high performance. The antibodies are also tested to ensure that endotoxin levels are low and that there is minimal cross-reactivity.
What information can you get from the supplier? A good supplier will have datasheets available that include information like tested applications and species as well as recommendations for success, such as starting concentrations and incubation times for antibody optimization. It is also important for the supplier to share data examples as well as citations of the product’s use in the literature.
Both monoclonal and polyclonal antibodies have advantages and disadvantages, and your specific target will determine which is better for you.
Monoclonal antibodies are produced by a single B cell clone. They are a homogenous population that binds with high affinity and specificity to a single epitope on a protein.
Polyclonal antibodies are produced by multiple B cells. They are a heterogeneous population that binds to multiple epitopes on a protein.
Monoclonal antibodies are more specific. If you need to distinguish between two highly related proteins, monoclonals may be the right choice. However, monoclonal antibodies are also more susceptible to “epitope masking,” meaning that the specific epitope may not be accessible at all times due to, for example, conformational changes in the protein. Epitope masking results in a false negative—the protein will not stain even though it is present in the sample. Polyclonal antibodies, on the other hand, bind to multiple epitopes and give a more robust signal. They are less likely to result in false negatives, and they’re more stable over a range of pH and salt concentration. However, they are also more likely to give a higher background signal.
When selecting an antibody, it is highly important to look at products that have been validated in your particular application. Just because an antibody works well for IHC, for example, does not mean that it would be appropriate for Western blot. Both of these applications require different types of sample prep, which could potentially result in staining differences. If you do choose to use an antibody for an application that it has not been validated for, it is important to do careful controls to make sure that your results are accurate.
Even if you have selected the perfect antibody for your experiments, your results may still be unsatisfactory if the staining protocol has not been optimized for your sample. The quality of staining can be affected by the blocking solution, primary and secondary antibody concentrations, incubation time, temperature, etc. It is good practice to always titrate your antibody to find the optimal signal-to-noise ratio for your specific product: increasing the concentration will increase signal and noise, while decreasing the concentration will decrease signal and noise. Other ways to decrease nonspecific staining are to dilute the secondary antibody, increase the concentration of the blocking agent, and conduct long incubations at 4°C.
Choosing an antibody may be daunting, but your research depends on you getting it right. If you need help, vendors like R&D Systems have support teams available that can help direct you to appropriate products.
R&D Systems offers over 13,000 antibody products.