Antibody conjugation is a critical step in many molecular biology research assays. A number of factors contribute to successful conjugation, including the characteristics of the antibody itself and buffers or preservatives that may be present along with the antibody sample. Some researchers choose to conjugate their own antibodies, either to save money or because they do not find commercially available conjugated products to suit their needs. The “do-it-yourself” (DIY) approach also gives the user complete flexibility over which labels and colors to use with which antibodies.
Start with the right antibody
Not all antibodies are readily conjugable. Commercially available antibodies are often supplied in buffers such as BSA or sodium azide, either of which can compromise or prevent effective labeling. Antibodies in these types of solutions are suitable for Western blot, immunohistochemical/immunocytochemical staining and flow cytometry but often are difficult to conjugate. To address this challenge, some suppliers package antibodies without these preservatives. It’s also important to confirm at the outset that antibody quantity and concentration are sufficient for effective conjugation.
Decide on primary or secondary
Before embarking on conjugation, researchers must assess whether they will work with a conjugated primary or secondary antibody. Labeled secondary antibodies are easier to find. Their main advantage is that they amplify the signal; but on the downside, they may compromise the required specificity. Additionally, same-species primary antibodies cannot be used in some types of assays, such as colocalization studies, because the secondary can’t differentiate between the two targets. Conjugated primary antibodies often are preferred because of their more specific binding ability. Also, when using a labeled primary, incubating with a secondary is not required. To maintain cell viability, an antibody against an extracellular epitope must be used when imaging or sorting live cells.
DIY conjugation
Although it is certainly feasible and possible for researchers to conjugate their own antibodies, it is not a simple job. Those who choose to do so should be prepared for a painstaking process and allow ample time for each step—plus time for trial and error. As previously mentioned, conjugation should not be attempted if the sample contains BSA or sodium azide. Even though there are kits on the market that claim to enable conjugation in the presence of BSA or sodium azide, your time may be better spent choosing an already-available labeled primary antibody.
When choosing fluorophors, it’s important to avoid overlapping emission spectrums if colocalization of two different proteins is the purpose for labeling the antibodies. Following the steps and reading the fine print provided with manufacturers’ labeling kits is a good start, along with mapping out troubleshooting steps in advance. There are multiple variables that can affect successful conjugation, and many of them may need to be adjusted during the process. Troubleshooting can take hours or even days. In some cases, the chosen antibody may lose its binding abilities and/or binding specificity after labeling because of structural and physical issues, indicating the antibody simply is not conjugable.
Commercial preconjugated antibodies
Because of the tremendous amount of trial and error associated with DIY conjugation, more researchers are turning to commercial vendors for antibodies and conjugates. The selection available has grown rapidly. A vendor of preconjugated antibodies will have already gone through all the verification and quality-control steps to ensure that the product is ideally suited for various research applications, saving the researcher valuable time. When choosing to purchase a conjugated product, researchers should make sure the vendor has tested the labeled antibody in immunohistochemical and/or immunocytochemical staining or flow cytometry to ascertain that the antibody works.
Looking for published scientific papers citing the use of a specific labeled antibody can give the researcher the peace of mind that the antibody works and increase confidence in the antibody vendor. Also, researchers should ask for samples. Any reputable and trustworthy vendor will gladly supply a free or discounted sample. It is also possible that for certain disciplines, such as ion channel research, conjugated antibodies may be hard to find. So a good first step is to find an antibody vendor that specializes in tools for the research discipline of interest.
Immunohistochemical staining of rat dorsal root ganglia (DRG) frozen sections using Anti-NMDA Receptor 2B (NR2B) (extracellular)-ATTO-594 antibody from Alomone Labs. Staining is present in neuronal cell bodies. Hoechst 33342 is used as the counterstain (blue).
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Melanie Grably, Ph.D, Alomone Labs
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