How to Elute Biotinylated Proteins and Nucleic Acids from Streptavidin Beads (2025)

Streptavidin is a bacterial protein that binds to the small molecule biotin with extremely tight affinity; this is one of the strongest non-covalent interactions between a natural protein and its ligand (Sano et al, 1998). This powerful interaction has made streptavidin a very useful biotech tool for immobilizing and purifying biotinylated molecules such as other proteins or nucleic acids.

The flip side of this incredibly strong interaction is that it is very difficult to get your biotinylated molecule eluted off of streptavidin beads once it is bound, and harsh elution conditions are required.

Eluting biotinylated proteins and DNA from streptavidin beads requires harsh conditions like enzymatic digestion, extreme pH, organic solvents, and heat with excess biotin. Deciding which elution conditions to use will depend on your specific biotinylated molecule and its downstream purpose.

In this article, we’ll discuss the different methods researchers use to elute biotinylated molecules from streptavidin beads (Figure 1).

Figure 1. Biotinylatedproteins bind to streptavidin conjugatedto an agarose bead.

Article Table of Contents:

How to elute biotinylated proteins?

Enzymatic digestion

Acidic buffer

Detergent and heat

How to elute biotinylated nucleic acids?

Basic buffer and heat

Pure water and heat

Extraction with organic solvents

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References

How to elute biotinylated proteins

Proteins are biotinylated when a biotin molecule is covalently attached to the side chain of a lysine. You can specifically biotinylate a single lysine residue in an avi-tag using an enzyme (Figure 2, left) (Fairhead & Howarth, 2015). Alternatively, you can redundantly biotinylate many lysine residues throughout the protein with a chemical reaction (Figure 2, right) (Kay et al, 2009).

Figure 2. Specific vs redundant biotinylation of proteins. Proteins can either be specifically biotinylated on an avi-tag (left), or redundantly labeled on lysine residues throughout the protein (right).

The tight interaction between streptavidin and biotin means that biotinylated proteins can be purified or immobilized with high specificity using this interaction. Common elution methods for biotinylated proteins include:

• Enzymatic Digestion
• Acidic Buffer
• Detergents and Heat

Enzymatic digestion

One way to get biotinylated proteins off of streptavidin beads is to leave the biotin-streptavidin interaction intact, and cleave the rest of the protein off of the biotinylated bit. This can be achieved by specifically cleaving a linker between a biotinylated tag and the rest of the protein, or by chopping the protein into lots of little bits with a non-specific protease (Figure 3).

Figure 3. Cleavingproteins off of streptavidin beads. For specific biotinylation, thebiotinylated tag can be cut off with a specific protease (gray scissors),releasing the intact protein from the streptavidin beads (left). For redundantbiotinylation, non-specific proteases (pink scissors) cut the protein into abunch of small pieces (right). The pieces without biotinylation will elute fromthe streptavidin beads.

Specifically cleaving a linker between a biotinylated tag and the rest of the protein will elute a full-length and functional protein that could be used for lots of different purposes. In contrast, cleaving the bound proteins into lots of little bits will elute lots of peptide fragments. This latter approach is popular when using mass spectrometry for protein analysis because the little peptide pieces are still useful for protein identification. For other purposes, where you desire an intact protein, cleaving with a specific linker, or using one of these next elution approaches would be the better choice.

Acidic buffer

Extremely acidic buffers disrupt the interaction between biotin and streptavidin and are used to elute biotinylated proteins. Examples of acidic elution buffers include:

• 6M guanidine HCl
• 0.1M acetic acid
• 0.1M glycine HCl

These acidic buffers are also detrimental to the overallfold and activity of most proteins you will be eluting, so you’ll want toadjust the pH back to neutral levels as soon as possible. Concentrated Tris or other bases can be used forthis purpose, and should be addedto the elution as soon as possible.

Detergent and heat

Lastly, detergent and heat are used to disrupt the biotin-streptavidin interaction and elute biotinylated proteins from streptavidin beads.

A harsh example of this is adding SDS-PAGE loading buffer to the streptavidin beads, boiling the sample, then loading the supernatant onto a gel. This is typically used when running the proteins on an SDS-PAGE gel is the intended downstream use, and is sufficiently harsh so that streptavidin elutes from the beads, in addition to the biotinylated proteins. For more information about SDS-PAGE – check out this article.

One research group set out to develop a milder detergent and heat elution technique such that only biotinylated proteins, but not streptavidin, would elute. This would be useful in applications where streptavidin contamination is detrimental – such as detecting scarce proteins. Their optimized elution buffer was: 0.4% SDS, 1% IGEPAL-CA630 (a detergent), and 25mM biotin with heating at 95 C for 5 minutes (Cheah and Yamada, 2017). For comparison, SDS-PAGE loading buffer contains 2% SDS.

An additional advantage of this technique is that streptavidin is still active, meaning that streptavidin beads can be reused a few times with careful benchmarking. In contrast, streptavidin beads are destroyed by more stringent elution techniques, like non-specific enzymatic digestion and SDS-PAGE loading buffer, and cannot be reused for additional purifications.

How to elute biotinylated nucleic acids?

Ok, now that we’ve discussed eluting biotinylated proteins, let’s move on to how to elute biotinylated nucleic acids. Nucleic acids are often biotinylated to assist in purifying them for downstream applications such as DNA sequencing, and structural or functional analyses. Common methods for eluting biotinylated nucleic acids include:

• Basic buffer and heat
• Pure water and heat
• Extraction with organic solvents

Basic buffer and heat

Ammonium hydroxide has been used to elute biotinylated DNA from streptavidin at elevated temperatures with high efficiency (Jurinke et al, 1997). Unfortunately, this harsh method also damages the DNA bases. So this approach should be avoided in favor of the following methods for virtually all downstream applications.

Pure water and heat

Alternatively, biotin also efficiently dissociates from streptavidin in pure water at temperatures greater than 70 C. Unlike the harsh basic buffer described above, DNA bases are not damaged with this elution technique (Holmberg et al, 2005).

However, the presence of common buffer elements, such as salt and pH buffers, limit the efficiency of biotin elution with this method. This means that DNA harvested from most biological settings cannot be eluted this way, or will need to undergo an additional intermediate step of buffer exchange into pure water first.

Extraction with organic solvents

An additional limitation of eluting biotinylated nucleic acids with heat is that elevated temperatures will melt nucleic acid base pairing (Figure 4). For many downstream applications, such as sequencing, that is not a problem since these interactions would be disrupted later in the workflow anyway.

However, for interactions that need the native structure of the nucleic acids to be maintained, it is better to avoid elevated temperatures.

Figure 4. Heatdenatures nucleic acid base pairing. For downstream applications that requireretaining native nucleic acid structure, heat must be avoided during theelution step.

Phenol-chloroform extraction, which is canonically used to separate nucleic acids from complex biological mixtures (Sambrook and Russell, 2006), can also be used to elute DNA off of streptavidin beads. Any proteins in the sample, including streptavidin on the beads, will be denatured using this approach, so you won’t want to reuse agarose beads after performing the extraction. However, nucleic acid interactions and structure remain intact after the extraction, and the biotin is still functional if you need to use it again downstream of this elution (Bearden et al, 2019).

Those are many of the common methods for eluting biotinylated proteins and nucleic acids from streptavidin beads. It is worth carefully considering which method will work best for you depending on the type of molecule you’re working with, how it is biotinylated, and what the downstream purpose is for the biotinylated molecule.

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