When using streptavidin agarose beads to purify biotinylated nucleic acids or proteins, there are a number of different ways to elute your biotinylated molecules from the beads. We cover many of those elution techniques in this article.
One of those elution methods involves using free biotin to compete for binding to the streptavidin beads to release your biotinylated molecule of interest. So, to make this elution buffer you have to get biotin into solution. Biotin is not very soluble at high concentrations in neutral pH, water-based buffers. But luckily, knowing a few tricks will allow you to make your biotin solutions with ease!
To make highly concentrated stock solutions of biotin, add either a strong base, such as sodium hydroxide, or an organic solvent, such as DMSO, to coax the biotin into solution. When this stock solution is diluted into your final buffer, the biotin will remain in solution.
In this article, we’ll touch on the general properties of solubility and how to solubilize biotin in concentrated stock solutions and into your final working buffers.
Article Table of Contents
Diluting you stock solution to get your final buffer
Let’s imagine a scenario in the lab. You’re purifying a biotinylated protein or nucleic acid using streptavidin agarose beads. To elute the biotinylated molecule off of the streptavidin beads, you’re trying to prepare an elution solution with biotin in it. No problem – you’ve made plenty of buffers before.
You pull the GoldBio biotin out of the fridge and calculate exactly how much you need to add to make your desired biotin concentration for the elution buffer. You add the biotin to your solution and put it on a stir plate to mix it up really well. Hmmm, the biotin doesn’t seem to be going into solution very fast.
No problem, you go check your email for a second to let the stir bar do its work. But when you come back that biotin powder still hasn’t gone into solution and is floating at the top of your buffer! What in the world is going on here?
Biotin is poorly soluble in neutral pH, water-based solutions (National Center for Biotechnology Information, 2025). Since most conventional biological buffers are around neutral pH and are water-based, biotin likely won’t solubilize in your desired buffer the way that sodium chloride would.
Don’t despair! There are a few simple tricks to get biotin into solution. But first let’s briefly cover what we mean by solubility in the first place.
Solubility
Solubility is essentially a measure of how well a molecule is mixed into a liquid solution. Oftentimes in biology, the liquid we’re referring to is essentially water with a few other things mixed in. Water is a polar molecule, which means it has a partial negative charge on its oxygen atoms and partial positive charge on its hydrogen atoms (Figure 1).
Figure 1. Water is a polar molecule with partial positive charges (d+) on the hydrogen atoms (H) and a partial negative charge (d-) on the oxygen atom (O).
Since water is polar, other polar and charged molecules will interact with the water molecules and be very soluble. Another way of saying this is that these molecules will be well mixed in a water solution. Before, when we mentioned that sodium chloride is very soluble in water this is because it dissociates into positive sodium and negative chloride ions that readily interact with water molecules (Figure 2).
Figure 2. Charged molecules like sodium (Na+) and chloride (Cl-) readily dissolve in water by interacting with the complementary partial charges on oxygen and hydrogen.
Biotin is basically the opposite in terms of its chemical properties. Biotin is hydrophobic, which means “water avoiding.” Biotin would rather clump together and interact with itself rather than with water molecules (Figure 3).
Making stock biotin solutions
With biotin, it’s easiest to first make a concentrated stock solution where you solubilize the biotin, then dilute the stock solution into your final purification buffer.
When making a stock biotin solution, I typically want it to be at least 10 times more concentrated than the concentration I desire it to be in my final buffer. So, for example, if my elution buffer needs 20mM biotin, then I’ll make a stock solution of at least 200mM biotin.
To make the concentrated biotin stock solutions, you have a couple of options:
- Make your solution slightly basic
- Add an organic solvent like DMSO
- Heat the biotin solution
Adding base
For full disclosure, this is my preferred method for solubilizing biotin. Add the biotin to water and add a stir bar. As we discussed above, the biotin will not go into solution yet and will mostly float around on the top of the liquid. Carefully, add concentrated sodium hydroxide, or another strong base, just a drop or two at a time. The buffer doesn’t need to be very basic, so it usually takes just a few drops to solubilize the biotin. You don’t want to add too much base, otherwise your stock biotin solution will dramatically change the pH of your final buffer when you add the biotin to it, as we discuss below.
It is not crucial to measure the pH of your stock solution. It will, however, be important to measure the pH of your final buffer after you’ve added the stock biotin solution to it, to make sure you’re not changing the pH so drastically to damage the biomolecules you’re purifying or disrupt downstream experiments. We’ll discuss this more in the final buffer section below.
Why does base solubilize biotin? Making the buffer more basic fully deprotonates the carboxylic acid on biotin, making biotin negative (Figure 4). Charged ions interact better with polar water molecules.
Figure 4. Biotin
becomes negatively charged at basic pH when a positive hydrogen atom
dissociates from its carboxylic acid moiety.
Adding organic solvent
Biotin is very soluble in organic solvents such as DMSO (National Center for Biotechnology Information, 2025). That’s because these solvents are more hydrophobic than water, so they are better at interacting with hydrophobic biotin molecules (Figure 5).
Figure 5. Biotin
is more soluble and intermixed with solvent in a DMSO-water mixture.
So, for your stock solution, you can solubilize biotin in a 10% DMSO (90% water solution). Of course, you can use a higher percentage of DMSO, and that will help solubilize biotin. But you want to make sure that when you dilute the biotin into your final solution, that the final concentration of DMSO is compatible with the biological molecules you are trying to purify, and with the downstream experiment you’re purifying those molecules for.
Heating the solution
Lastly, if you just heat your biotin stock concentration, it will go into solution. I like this method the least because it takes longer to heat and cool your solution, compared to the above techniques. But if you don’t want to worry about checking the pH of your final solution, or having too much organic solvent present, then this is an alternative option.
Another reason I don’t prefer this approach is that often, after your stock solution has cooled some of the biotin will precipitate back into a solid form. So if you’re using it right away, that’s fine. But, if you’re storing the stock solution for later, you will likely need to redo the heating/cooling cycle each time you use your biotin stock, which adds to the time-consuming nature of this approach.
Why don’t the other two approaches also suffer from biotin reversibly precipitating? Well, you’re maintaining the condition of the stock solution that keeps biotin in solution – either basic pH or organic solvent. It’s only once your biotin is diluted that this condition is changed. But at that point, the concentration of biotin is lower so it will stay in solution. With the heating/cooling method, you’re removing the condition (heat) that keeps biotin in solution, while the biotin concentration is still high.
Diluting you stock solution to get your final buffer
Now comes the easy part – just dilute your stock solution into your final buffer. Now that biotin is at a lower concentration, it should stay in solution just fine.
The only thing to make sure of here is that adding the biotin didn’t significantly change the final buffer in a way that will impact the downstream use.
What do I mean by that? Well, if you used the minimal amount of sodium hydroxide to solubilize the biotin, and your biotin stock solution is only slightly alkaline, then diluting it into your final buffer will likely have a very small change on the final buffer’s pH, or perhaps no change at all. But just to be sure, check your final buffer’s pH after adding biotin to make sure this is the case.
If the biotin stock solution did significantly change the buffer’s pH, you can always use a few drops of concentrated acid to lower the pH back to the original value.
If instead of sodium hydroxide you used DMSO, then just be sure that the final concentration of DMSO isn’t impacting your purification or downstream experiments. Typically, most proteins tolerate DMSO concentrations up to 5% just fine, but you can always use a functional assay or other quality control to ensure that your protein still functions in the percentage of DMSO that’s in your elution buffer.
That’s all there is to it - what started out as a frustrating lesson in solubility, or the lack thereof, is easily fixed with a little base or organic solvent. Now, you’re ready to elute those biotinylated biomolecules!
