We all need a helping hand sometimes. Especially when the heat gets turned up, getting some aid can make all the difference between success and failure.

These sentiments are just as true in the molecular world as they are in our everyday lives. Enzymatic assays, especially those under elevated temperature, can often use some help to catalyze their reactions. That’s where BSA comes in.

Bovine serum albumin (BSA) is frequently used as a stabilizing agent in enzymatic reactions. BSA both stabilizes many enzymes, especially at high temperatures, and prevents adsorption of enzymes onto surfaces.

Here, we’ll discuss why BSA stabilizes enzyme reactions, and how it is used in different biochemical experiments.

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In this article:

How BSA stabilizes enzymes

BSA also prevents adsorption of enzymes

Experiments to include BSA in

When to use a different type of stabilizer/blocking agent

Which type of BSA to use as a blocking agent

References

 

How BSA stabilizes enzymes

Relative to the crowded intracellular environment, a test tube can be a rather lonely and vacuous environment. Indeed, in vitro experiments are kind of a foreign environment compared to the spaces that proteins normally occupy. Model proteins, like BSA, can be used to mimic molecular crowding more akin to the cellular environment (Protter et al, 2018).

BSA isn’t always just a passive space filler, however, it can also play active roles in stabilizing enzymes. This just means that BSA helps preserve the enzyme’s structure to better maintain its biological activity over time. Insults, such as high temperature enhance enzyme denaturation, and BSA counteracts that process.

BSA has a relatively hydrophobic surface. So, compared to polar aqueous buffers, the hydrophobic surface of BSA can interact with hydrophobic surfaces on enzymes, thereby stabilizing them (Figure 1).

How exactly does BSA stabilize enzymes? Let’s consider the old adage “oil and water don’t mix”. The hydrophobic parts of proteins are like oil, and they would rather interact with each other than with water molecules or the salt ions in your buffer.

Figure 1. BSA (gray oval) binds to and stabilizes a protein of interest (blue triangle) via surface hydrophobic patches (orange ovals on each protein). These hydrophobic patches huddle together to minimize unfavorable interactions with water molecules and salt ions.

 

This stabilization is particularly apparent at higher temperatures. Proteins start to partially unfold at elevated temperatures, revealing even more hydrophobic protein surfaces which were buried on the inside of the protein at lower temperatures. These newly exposed hydrophobic parts will readily bind to hydrophobic BSA, which again is where the stabilizing impact of BSA comes from (Chang and Mahoney, 1995) (Figure 2).

Figure 2. BSA is even more stabilizing at higher temperatures when additional hydrophobic patches may be exposed on your protein of interest.

By the way, we have an article about how proteins are purified based on their hydrophobicity, so if you’re interested in learning more about protein hydrophobic interactions that is a great place to start.

 

BSA also prevents adsorption of enzymes

BSA or other blocking agents can make great additives for enzymatic assays. In some cases, BSA really is stabilizing the enzyme through the means that we just discussed. But in other scenarios the benefit of including BSA is that it will coat plastic pipette tips, centrifuge tubes, slides, membranes, or whatever other surfaces your sample will touch that non-specifically bind to protein.

If your precious enzyme is binding to those surfaces it will likely be inactive which will dampen the overall activity of your reaction. For example, you might only have 50% of the enzyme that you think you have because the rest is stuck on a surface where it can’t catalyze its reaction (Figure 3, left). By adding BSA you coat those surfaces keeping your enzyme in solution and active (Figure 3, right).  

Figure 3. Left, without BSA some enzymes (blue triangles) will adsorb onto plastic surfaces rendering them inactive. Right, BSA (gray ovals) adsorbs onto the tube keeping the enzymes in solution and active.

 

That’s why BSA, or other blocking agents, are often included in enzymatic reactions. Ultimately, if your reaction is working well with BSA included in the mix you may not really care whether the BSA is helping by stabilizing your enzyme, serving as a blocking agent, or both. If your reaction is not broken, don’t fix it!

 

Experiments to include BSA in

BSA is widely used in all kinds of enzymatic reactions. Common examples include PCR and restriction digests of DNA. Stabilization using BSA is really helpful for these kinds of reactions since the reactions take place at elevated temperatures.

Thinking more broadly about a variety of enzymes, it is quite common to incubate the reaction at the “natural” temperature for the host cell or organism from which the enzyme was derived. So, for human proteins (or for bacterial proteins where the bacteria live in humans) that temperature is usually 37 °C. For yeast (Saccharomyces cerevisiae), the default temperature for common lab variations is 30 °C, though the optimal temperature will change if you’re working with different strains (Salvadó et al, 2011).

This temperature variance is because enzymes are often tuned to function optimally, or most efficiently at the temperature that they normally exist at (Feller, 2003; Klinman & Kohen, 2014; Nguyen et al, 2017). For such enzymatic reactions, the stabilization provided by BSA is especially helpful at elevated temperatures, compared to room-temperature or 4 °C reactions.   

BSA is also a great additive to a lot of biochemical experiments, not just those involving enzymes. Its role as a blocking agent makes BSA useful for including for all kinds of in vitro experiments including: western blots, ELISAs, immunohistochemistry and immunofluorescence, EMSAs, and more. For any experiment where your precious protein(s) will be exposed to sticky lab surfaces such as centrifuge tubes, pipette tips, membranes, and more, it is worth including a blocking agent such as BSA.

We cover some of these experiments, and the purpose of BSA as a blocking agent, in more detail in this article, so check it out if you want to learn more.

 

When to use a different type of stabilizer/blocking agent

BSA is a very popular choice as an enzyme stabilizer and blocking agent, but it is not the only option. In certain experiments other stabilizers/blocking agents could be better choices.

For example, I was optimizing a protease cleavage assay. A protease is an enzyme that cuts up other proteins into smaller pieces. In this assay I was using an SDS-PAGE gel for reading out enzyme activity, and I could tell from my gels that the substrate protein was varying more than it should between experiments, suggesting that the protein was sticking to the pipette tips, centrifuge tubes, or something else it was coming in contact with during the experiment.

This could be an example where BSA would be a good choice as a blocking agent. However, the molecular weight of the substrate protein was very similar to BSA, so if I used BSA in my reaction it would mask the uncleaved protein. So, I used a detergent instead and that worked well for providing more reproducible results without any interference in the SDS-PAGE gel readout.

This example, however, is one of the rare exceptions where it makes sense to go with another stabilizer or blocking agent instead of BSA. In most of the experiments listed above, BSA is a helpful and invisible bystander. If BSA doesn’t interfere with the signal in your experiment, then I would recommend trying it first.

Which type of BSA to use as a blocking agent

There are lots of different varieties of BSA, so it’s important to know which type to use based on which experiment you’re doing.

For the purposes of enzyme stabilization, however, the truth is that pretty much any type of BSA will work. One important feature is that you’ll want to make sure that your source of BSA is protease free, such as the following products:

·         Bovine Serum Albumin (BSA), Fraction V, Protease Free

·         Bovine Serum Albumin (BSA), Cohn Analog Grade

·         Bovine Serum Albumin (BSA), Standard Grade pH 7.0 (NZ)

Protease contamination would degrade your enzyme, so you’ll definitely want to avoid that.

The only other thing to keep in mind is what type of enzyme you’re working with. If fatty acids will interfere with your experiment – for example if your enzyme is a fatty acid binding protein – then you would probably want to use one of our versions of BSA that doesn’t have any fatty acids in it such as:

·         Bovine Serum Albumin (BSA), Fraction V, Fatty Acid Free for tissue culture

·         Bovine Serum Albumin (BSA), Precision Grade Fatty Acid Free (NZ)

 

Ultimately, that’s how BSA can enhance enzymatic reactions, and other biochemical experiments, by stabilizing enzymes, serving as blocking agent, or both. If BSA, or another blocking agent is working reproducibly in your experimental setup, you probably don’t care too much how BSA is helping – just keep cranking that data out. GoldBio has a variety of affordable and high-quality BSA research reagents. Check out the links below to learn more about those products.

 

References

Chang, B. S., & Mahoney, R. R. (1995). Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions. Biotechnology and applied biochemistry, 22(2), 203–214.

Feller G. (2003). Molecular adaptations to cold in psychrophilic enzymes. Cellular and molecular life sciences : CMLS, 60(4), 648–662. https://doi.org/10.1007/s00018-003-2155-3

Klinman, J. P., & Kohen, A. (2014). Evolutionary aspects of enzyme dynamics. The Journal of biological chemistry, 289(44), 30205–30212. https://doi.org/10.1074/jbc.R114.565515

Nicoya Lifesciences Inc. (2020, December 10). 4 ways to reduce non-specific binding in surface plasmon resonance experiments. https://nicoyalife.com/blog/4-ways-reduce-non-specific-binding-spr/

Nguyen, V., Wilson, C., Hoemberger, M., Stiller, J. B., Agafonov, R. V., Kutter, S., English, J., Theobald, D. L., & Kern, D. (2017). Evolutionary drivers of thermoadaptation in enzyme catalysis. Science (New York, N.Y.), 355(6322), 289–294. https://doi.org/10.1126/science.aah3717

Patsnap. (2025, April 29). Why add BSA to PCR mixes? The science behind stabilization. Synapse. https://synapse.patsnap.com/article/why-add-bsa-to-pcr-mixes-the-science-behind-stabilization

Protter, D. S. W., Rao, B. S., Van Treeck, B., Lin, Y., Mizoue, L., Rosen, M. K., & Parker, R. (2018). Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly. Cell reports, 22(6), 1401–1412. https://doi.org/10.1016/j.celrep.2018.01.036

Salvadó, Z., Arroyo-López, F. N., Guillamón, J. M., Salazar, G., Querol, A., & Barrio, E. (2011). Temperature adaptation markedly determines evolution within the genus Saccharomyces. Applied and environmental microbiology, 77(7), 2292–2302. https://doi.org/10.1128/AEM.01861-10

 

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BSA protein purification Simon Currie

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