Covalently conjugating a small molecule to an antibody’s surface is a process called antibody “labeling.” Labeling antibodies with small molecules such as biotin or fluorophores provides a convenient handle for visualizing and isolating the antibody.  

Scientists label antibodies with molecules like biotin or fluorophores to visualize, quantify, and isolate antibodies and the antigens that they bind to. These tools enable scientists to examine particular proteins in molecularly complex samples such as biochemical mixtures, cells, and tissues.

In this article we’ll discuss why you might want to label your antibodies, the differences between two common labels: biotin and fluorophores, and look at many examples of the types of experiments and cutting-edge research that is enabled with labeled antibodies.

Table of Contents:

Antibody labeling

Biotin vs fluorophore

Experiments that use labeled antibodies

Binding interactions

Western blot

Immunohistochemistry & immunofluoresence

Microscopy and imaging

Flow cytometry

References

 

Antibody labeling

Two of the most common ways to label an antibody are to conjugate a fluorophore or biotin to the antibody surface (Figure 1).

Figure 1. Antibodies are labeled with a fluorophore (left) or biotin (right).

 

Adding a fluorophore to the antibody allows you to directly monitor the antibody using fluorescence. We’ll discuss some of the key experiments where having a fluorescent antibody is useful later in this article.

In contrast, labeling with biotin enables you to pull down the antibody with streptavidin, or to indirectly monitor the antibody using a particular streptavidin conjugate. For example, streptavidin conjugated to horseradish peroxidase (HRP) is used to visualize biotinylated antibodies.  

 

Biotin vs fluorophore

In some of the experimental applications listed below, you could use either a biotinylated antibody or an antibody labelled with a fluorophore. So, before we get into those specific experiments, let’s discuss the general differences between antibodies that are labelled with biotin or fluorophore.

You will get more sensitivity in detecting low concentration antibodies when they’re labelled with biotin. So, if you are trying to detect something that is very rare, then biotin labelling would be the appropriate choice (Figure 2).

Conversely, while fluorescence detection is not as sensitive as biotin, it is a better choice for quantitative analyses. This is because fluorescence has a wider linear range where if you double your labeled antibody, the fluorescence intensity (light emitted) will also double (Figure 2).

Figure 2. Fluorophore labeling is less sensitive but more quantitative (left), whereas biotin labeling is more sensitive but less quantitative (right). 

 

Even fluorophores do not have an infinite linear range, so if you’re using them for quantification then it’s important that you determine the linear range with your experimental setup (Currie et al, 2023). By the way, if you want to brush up on the basics of fluorescence, this is a great article for that.

To summarize, biotin is usually the way to go if you just need a binary yes/no answer on whether something is present, whereas fluorescence will give better quantitation if you want to know how much of something is there.

Another difference is that there is a broad range of fluorophores with different excitation and emission spectra. This means that you could simultaneously monitor multiple different antibodies using distinct fluorophores on each. Having multiple labels allows you to track multiple molecules at the same time in the same experiment, for example to see if two different proteins colocalize to the same location in a cell. Later in this article we’ll see how multiple labels helps differentiate between colorectal cancer cells and gut microbes.

You will not have that flexibility to monitor different antibodies with biotin, though the signal from HRP is more stable over time as it is not susceptible to photobleaching.

Ok, with those general differences in mind, let’s get to different applications for labeled antibodies.

 

Experiments that use labeled antibodies

Labeling antibodies with biotin or a fluorophore is really useful for detection, quantitation, and measuring interactions with other molecules. Let’s go over a variety of experiments that researchers use labeled antibodies for.

 

Binding interactions

Biology is a contact sport! Interaction between different molecules is the foundation of all biological activities including metabolism, cell division, signaling pathways, and much more. As such, understanding which molecules interact with each other, and how strong those binding interactions are, is an important cornerstone for understanding biology.

Adding a biotin functionalizes an antibody for measuring binding interactions using assays such as Enzyme Linked Immunosorbent Assay (ELISA), surface plasmon resonance (SPR), and streptavidin pull-downs. 

One research group biotinylated antibodies that bind to bacterial cells using the Mix-n-Stain^TM Biotin Antibody Labeling Kit (Shi et al 2018). This allowed them to use SPR to measure the affinity, or how tightly the antibodies bound to bacterial cells. They also used the biotinylated antibodies in optical tracking experiments, which are single molecule experiments that track the movement of an individual antibody. They found similar binding affinities in both SPR and optical tracking experiments.

While not everyone will have access to advanced optical traps, biotinylating molecules is a powerful approach for measuring binding affinities through a variety of experimental methods. 

 

Western blot

Western blotting traditionally involves a primary antibody that recognizes your protein of interest, and a secondary antibody that recognizes the primary antibody and produces a chemiluminescent signal (Figure 3, left).  

Figure 3. Traditionally Western blots use a primary antibody (orange) to recognize the antigen (blue protein) and a secondary antibody (purple) conjugated to HRP (gray) to generate a chemiluminescent signal (left). Using a fluorophore-labeled primary antibody (orange) enables direct detection, saving time and making the blot more quantitative (right).

Alternatively, by labeling your primary antibody with a fluorescent dye you can skip the second incubation and wash steps which saves time in the lab (Figure 3, right).

Additionally, using a fluorescent dye instead of chemiluminescence has advantages such as a wider linear range making the Western blot more quantitative (Eaton et al, 2014). So, by using a labeled antibody you get a more quantitative experiment in less time!

 

Immunohistochemistry & immunofluorescence

Immunohistochemistry and immunofluorescence are techniques for using a labeled antibody to visualize molecules in tissue sections (immunohistochemistry) or individual cells (immunofluorescence).

There are around 400 distinct cell types in the human body, such as blood cells, liver cells, heart cells, and including all of the different subtypes that make up each of these different organs (Hatton et al, 2023). Different cell types express distinct proteins on their cell surface. These proteins are used as “markers” to identify the cell type using antibodies that bind to the distinct markers.

For example, in one effort to develop a better cell culture model of colorectal cancer, researchers used fluorophore-conjugated antibodies to detect two markers of colorectal cancer cells: carcinoembryonic antigen (CEA) and C2 antigen (Arul et al, 2014).

They were able to detect both antigens simultaneously by using a green fluorophore (Mix-n-Stain^TM CF® 488A) on the CEA-recognizing antibody and a red fluorophore on the C2-binding antibody (Mix-n-Stain^TM CF® 594) (Figure 4). So, this is an example where it was really useful to  use fluorophores instead of biotin on the antibodies. These tools helped them optimize a protocol to isolate colorectal cancer cells from patient biopsies while removing microbes that reside within the colon which do not have the markers recognized by the CEA and C2 antibodies.

 

Microscopy and imaging

Fluorescent molecules are powerful tools that provide dynamic and beautiful images through microscopy and other methods. Note that fluorescence microscopy and immunofluorescence can mean the same thing when you’re using fluorophore labeled antibodies for visualization. However, each of these terms can be distinct depending on which reagents and exact experimental setup you’re using.

One research group used an antibody labeled with a far-red dye (Mix-n-Stain^TM CF® 640R) to label neurons during live cell imaging (Guardia et al, 2019). Using a clever experimental setup with biotinylated proteins they were able to control organelle movement and positioning within neurons,  yet another example of the power of biotinylating proteins.

Another group labeled purified proteins with an orange fluorescent dye (Mix-n-Stain^TM CF® 555) and used microscopy to visualize in vitro mixtures of protein complexes (Trivedi et al, 2019). Their experiments identified the subset of proteins capable of forming centromeres, which are the protein complexes that bind to mitotic chromosomes and pull them apart during cell division. Importantly, this example demonstrates that antibody labeling kits can be applied to other types of proteins, though a little additional optimization and quality control may be necessary.

 

Flow cytometry

Flow cytometry is a method for sorting cells in a population based on how much of a particular protein they have on their surface. By labeling antibodies with a fluorophore you can separate and count how many cells have a lot of a certain protein versus how many cells are lacking that molecule.

One study was investigating the molecular mechanisms of how the human immunodeficiency virus (HIV) kills human immune cells, and they used flow cytometry to help figure this out (Sainski et al, 2014).

They labeled an antibody that binds to the HIV protein Casp8p41 with a far-red fluorescent dye (Mix-n-Stain^TM CF® 640R), and also labeled an antibody that recognizes the active conformation of the pro-apoptotic protein Bak with an orange fluorescent marker (Mix-n-Stain^TM CF® 532). Apoptosis is the process of cell death that HIV triggers in human immune cells.

This experiment showed that peripheral blood mononuclear cells from patients that had the HIV protein Casp8p41 also had higher levels of active Bak, indicating these cells were undergoing apoptosis.

So, essentially the HIV protein is hijacking the human cells’ natural apoptotic signals to kill them. These cellular results supported and provided clinical relevance for a lot of previous biochemical data the researchers had generated describing the interaction between Casp8p41 and Bak (Sainski et al, 2014).

 

Now that is a lot of cool science highlighting the power of labeling antibodies, and other proteins, with biotin and fluorescent dyes! If you’re ready to label antibodies, check out the rainbow of kits available at GoldBio below, as well as additional resources to give you the know-how to get your experiments right the first time.

 

 

References

Arul, M., Roslani, A. C., Ng, C. L., & Cheah, S. H. (2014). Culture of low passage colorectal cancer cells and demonstration of variation in selected tumour marker expression. Cytotechnology, 66(3), 481–491. https://doi.org/10.1007/s10616-013-9600-4

Biotium. (n.d.). Protocol: Fluorescent Western blotting. https://biotium.com/tech-tips-protocols/protocol-fluorescent-western-blotting/

Currie, S. L., Xing, W., Muhlrad, D., Decker, C. J., Parker, R., & Rosen, M. K. (2023). Quantitative reconstitution of yeast RNA processing bodies. Proceedings of the National Academy of Sciences of the United States of America, 120(14), e2214064120. https://doi.org/10.1073/pnas.2214064120

Eaton, S. L., Hurtado, M. L., Oldknow, K. J., Graham, L. C., Marchant, T. W., Gillingwater, T. H., Farquharson, C., & Wishart, T. M. (2014). A guide to modern quantitative fluorescent western blotting with troubleshooting strategies. Journal of visualized experiments : JoVE, (93), e52099. https://doi.org/10.3791/52099

Guardia, C. M., De Pace, R., Sen, A., Saric, A., Jarnik, M., Kolin, D. A., Kunwar, A., & Bonifacino, J. S. (2019). Reversible association with motor proteins (RAMP): A streptavidin-based method to manipulate organelle positioning. PLoS biology, 17(5), e3000279. https://doi.org/10.1371/journal.pbio.3000279

Hatton, I. A., Galbraith, E. D., Merleau, N. S. C., Miettinen, T. P., Smith, B. M., & Shander, J. A. (2023). The human cell count and size distribution. Proceedings of the National Academy of Sciences of the United States of America, 120(39), e2303077120. https://doi.org/10.1073/pnas.2303077120

Sainski, A. M., Dai, H., Natesampillai, S., Pang, Y. P., Bren, G. D., Cummins, N. W., Correia, C., Meng, X. W., Tarara, J. E., Ramirez-Alvarado, M., Katzmann, D. J., Ochsenbauer, C., Kappes, J. C., Kaufmann, S. H., & Badley, A. D. (2014). Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation. The Journal of cell biology, 206(7), 867–876. https://doi.org/10.1083/jcb.201405051

Shi, Y. Z., Xiong, S., Zhang, Y., Chin, L. K., Chen, Y.-, Zhang, J. B., Zhang, T. H., Ser, W., Larrson, A., Lim, S. H., Wu, J. H., Chen, T. N., Yang, Z. C., Hao, Y. L., Liedberg, B., Yap, P. H., Wang, K., Tsai, D. P., Qiu, C. W., & Liu, A. Q. (2018). Sculpting nanoparticle dynamics for single-bacteria-level screening and direct binding-efficiency measurement. Nature communications, 9(1), 815. https://doi.org/10.1038/s41467-018-03156-5

Trivedi, P., Palomba, F., Niedzialkowska, E., Digman, M. A., Gratton, E., & Stukenberg, P. T. (2019). The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex. Nature cell biology, 21(9), 1127–1137. https://doi.org/10.1038/s41556-019-0376-4

 

 

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