Everything About Labeling Proteins with NHS Esters

by Simon Currie

Handles are common in everyday life. We use them to help us open doors, cabinets, or to hold coffee mugs, cookware, and tools. They are used for all kinds of things.

There are handles in the molecular world as well that help us use and track proteins and other molecules. Molecular handles such as biotin and fluorophores are usually called “labels,” because they are covalently labeled onto the protein that they’re now bound to.

N-Hydroxysuccinimide (NHS) esters label proteins and peptides by reacting with amine functional groups on lysine residues. NHS esters conjugated to biotin or a fluorophore are frequently used to label proteins with these molecules thereby enabling downstream experiments.

In this article we’ll cover how NHS esters label proteins, how to label proteins, why you would want to label proteins, and finally some tips and tricks to keep in mind to make your labeling as successful as possible.

How NHS esters label proteins

NHS esters react with amine functional groups. In the context of proteins and peptides this means NHS esters will conjugate to the side chain of lysine residues (Figure 1).

Figure 1. Lysine residues form a covalent bond with NHS esters to label proteins with biotin (top) or fluorophores such as Cy3 (bottom).

Since lysine residues occur frequently in proteins, this means that labeling your protein with an NHS ester usually results in multiple lysine residues being labeled per protein. If you labeled your protein with a fluorophore, it’s possible to know the average extent of labeling for your sample by measuring the protein concentration and the fluorophore concentration after you’ve labeled your protein (Figure 2).

When labeling with biotin, you usually don’t worry about the exact extent of biotinylation, but rather just use a binary check to make sure that your protein is biotinylated at all. One easy way to do this is to see whether your biotinylated protein binds to streptavidin. GoldBio sells streptavidin-conjugated agarose beads that are perfect for this purpose.

This difference between streptavidin and fluorophores is because the fluorophores are detected directly and have a wider linear range of detection. Detecting biotinylation, in contrast, is more sensitive but gives less of a quantitative read-out of the extent to which the sample is biotinylated. This article delves into this difference between fluorophores and biotinylation in more detail.

Alternatively, you could use mass spectrometry for either biotin or fluorophore labeling to determine the extent of labeling.

Knowing the extent of labeling is not always crucial for downstream experiments. Oftentimes, it is good enough to know that your protein is labeled enough for you to detect it. However, if your experiment is quantitative in nature then it will be important to know the exact extent of labeling. For example, if you wanted to know how many protein molecules were in a cell or subcellular compartment, then you would need to know the extent of labeling (Trivedi et al, 2019).

labeled proteins and the extent of labeling

Figure 2. There will be a range of covalent labels per protein. With fluorescent labels you can determine the average extent of labeling by measuring the concentration of fluorophore and protein after removing unbound dye.

How to label your protein with an NHS Ester

Labeling proteins with NHS esters is a pretty easy and straightforward procedure. There are essentially five steps:

1. Prepare your protein for the labeling reaction. Typically, protein concentrations in the 1-5 mg/mL range work well. You’ll want to use an amine-free buffer such as PBS in the pH 7-9 range.

2. Immediately before use, dissolve the NHS Ester in DMSO to make a stock solution of 20 mM.

3. Add the NHS reagent to your protein sample so that the NHS is approximately 20-fold molar excess to the protein. This ratio can be changed depending on your desired extent of labeling and the specific protein being used.

4. Incubate the reaction for either: 30 minutes at room temperature, or 2 hours on ice.

5. Remove unreacted NHS reagent by dialysis or desalting.

That’s it, with just a few quick steps your protein is labeled with biotin or a fluorophore and ready for an experiment or to store until you’re ready to use it.

Experiments that use labeled proteins

Adding a biotin or fluorophore molecule to a protein is really helpful for detecting and quantifying that protein, as well as measuring interactions with other molecules. There are many experiments that use labeled proteins such as: assays measuring binding interactions, Western Blots, immunofluorescence and immunohistochemistry, advanced microscopy, and more.

For example, researchers used labeled proteins to generate a better cell culture model of colorectal cancer, and to investigate the molecular mechanisms behind HIV neutralizing human immune cells (Arul et al, 2014; Sainski et al, 2014).

We cover these examples, and others, in more detail in this article, which is focused on antibodies. However, the same experiments and reasons for labeling apply to proteins of all kinds, so check it out if you’re interested in learning more.

Troubleshooting tips and tricks

The abbreviated protocol we covered above makes labeling your protein seem really easy. However, just like any experiment, there are always little details that are very important to get right. Below, we cover many of these tips and tricks to be aware of when labeling with NHS esters.

· Do not use amine-containing buffers such as Tris or glycine in the labeling reaction.

Instead, use a non-amine-containing buffer in the pH 7-9 range such as PBS, HEPES, carbonate/bicarbonate, or borate.

· The extent of labeling will depend on the ratio of NHS reagent to protein of interest, and on the specific protein that is being labeled. If you get excess labeling, either reduce the molar excess of the NHS reagent, or choose a labeling reagent that targets a different reactive group. If your protein is not labeled, or barely labeled, increase the molar excess of the NHS reagent, and make sure that your protein has a surface-exposed lysine residue.

· If you have excessive background in your experiment, make sure that you sufficiently depleted free dye from your sample after labeling by dialysis or desalting.

· You may want to check your labeled protein in a functional or biophysical assay. NHS esters label rather indiscriminately on free lysine residues, so ensuring that the reaction doesn’t interfere with your protein’s function or binding to important partners is likely relevant for your downstream experiments. If labeling does interfere with protein function, reduce the extent of labeling or try a different type of labeling reagent.

Now that you know all about the power of NHS esters, it’s time to label your protein. At GoldBio we have reliable NHS reagents for biotinylating or labeling with the fluorophores Cy5 and Cy3. You can find those products below, as well as lots of great resources to help you efficiently label your proteins.

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

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

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