How Biotinylated Molecules are Purified with Streptavidin Beads

Streptavidin is a bacterial protein that binds to the small molecule biotin with extremely high affinity. This is one of the strongest noncovalent interactions ever discovered! Streptavidin agarose beads leverage the strength of this interaction to serve as a platform for purifying biotinylated biomolecules such as proteins and DNA, and for evaluating molecular interactions (Figure 1).

Biotinylated molecules are purified with streptavidin beads by using the strong biotin-streptavidin binding interaction. When mixed, biotinylated targets bind to the beads, allowing unbound molecules to wash away, isolating the desired protein or DNA.

Figure 1. Streptavidin agarose beads with a biotinylated protein bound.

Article Contents:

What are Biotinylated Molecules?

How are biotinylated molecules purified?

What else are biotinylated molecules used for?

Related Products

Related Resources

References

What are Biotinylated Molecules?

Biotinylated molecules are proteins or nucleic acids with the small molecule biotin covalently attached to them.

There are a few examples of proteins that are biotinylated in nature (Niers et al, 2011). But usually when we’re talking about biotinylated molecules in scientific research, we’re referring to molecules that scientists have intentionally biotinylated as a method for further studying those molecules.

For example, scientists can specifically biotinylate one particular lysine residue in an avi-tag using the enzyme BirA (Figure 2, left) (Fairhead & Howarth, 2015). An avi-tag is an affinity tag that can be added to your protein of interest in order to purify or immobilize it. Unlike other affinity tags, avi-tags require biotinylation to be useful in this context.

Instead of enzymatic biotinylation of a specific lysine residue, proteins can also be redundantly biotinylated on many lysine residues throughout the protein using a chemical reaction (Figure 2, right) (Kay et al, 2009). The advantages of this technique are that your protein doesn’t have to have an avi-tag on it, and you can biotinylate many different parts of the protein which can be useful in case you are going to later chop up your protein into bits, as we’ll discuss more in the next section.

Figure 2. Proteins can be enzymatically biotinylated on a specific lysine residue on an avi-tag (left), or chemically biotinylated on many different lysine residues throughout the protein (right).

How are biotinylated molecules purified?

If you’ve read our other articles on affinity tags or affinity purification of antibodies, then you already know that affinity purification involves three key steps: 1) binding the target protein to the beads, 2) washing away contaminating proteins, and 3) eluting your protein of interest (Figure 3).

Figure 3. Biotinylated molecules bind to streptavidin agarose beads (column 2). After washing, the biotinylated molecules are eluted a number of different ways , including with an acidic pH elution buffer that weakens the interaction between the streptavidin and biotin (column 3).

This general workflow is the same with biotinylated proteins and streptavidin beads. However, there are a few key things to know that make these steps a little bit different for the streptavidin-biotin pairing.

Remember when we mentioned how this interaction is one of the strongest natural interactions observed so far? The strength of this interaction means that you can stringently wash the beads and achieve better purity of your target protein compared to something like his-tag and nickel bead interactions.

The downside of the strength of this interaction is that it isn’t so easy to elute biotinylated molecules from streptavidin beads. Relatively harsh elution conditions are required such as buffers with extreme pH, organic solvents, heating the beads, or even cutting your molecule off of the beads.

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What else are biotinylated molecules used for?

Another example where it can be really great to have a biotinylated molecule is when you’re interested in finding out what other types of molecules it interacts with.

Say, for example, you’re studying a protein that causes cancer. Almost always in biology, molecules never act alone. Rather, they have molecular partners that aid them in carrying out their functions. And so, if you wanted to know more about how this protein causes cancer, one of the first things you would do is try to find other molecules that contribute to its oncogenic phenotype.

One way you could do this experiment is to biotinylate the cancer-causing protein, immobilize it onto streptavidin beads, and then find other proteins from cancer cells that bind to your protein (Figure 4). Once you discovered a protein, or proteins, that interact with the cancer-causing protein, there are other experiments you could do to figure out which of these interactions functionally contribute to oncogenesis.

Figure 4. After binding your biotinylated target protein to streptavidin beads, you can use this as a bait to search for interacting proteins.

This is particularly exciting because if you figured out a key protein-protein interaction that drives cancer, then you could try to develop a drug that stops cancer by preventing this protein-protein interaction. This exact method has been used to develop several existing drugs used by cancer patients today (Nada et al, 2024).

While this example focuses on proteins, the same overall method can be applied to DNA molecules. Say there is a single nucleotide change associated with cancer, but it isn’t known how this change in DNA sequence leads to cancer. You could biotinylate the DNA and use streptavidin beads to pull down interacting proteins.

A particularly clever way to do this would be to compare the original DNA sequence with the mutated cancer sequence to help determine which proteins might specifically be involved in the cancer function (Figure 5). Just as with the above example, this is the exact kind of experiment that scientists have done to figure out what is going wrong in certain types of cancer (Carrasco Pro et al, 2023).

Figure 5. You can use streptavidin beads to pull down interacting proteins against biotinylated DNA. In this case a blue protein interacts with both wild type and mutated DNA, whereas only the dark green protein interacts with the mutated DNA. In this hypothetical example, you would probably be interested in figuring out what the dark green protein is.

So, that’s all about how biotinylated molecules are purified and immobilized using streptavidin beads. Check out the resources below to learn more about related topics and GoldBio products that will aid you in executing the types of experiments we’ve mentioned in this article.