Have you ever needed to block out the background noise and focus on what’s truly important in your life? In the molecular world, bovine serum albumin (BSA) is a commonly-used blocking agent to prevent spurious or non-specific molecular interactions to focus on real and important interactions.

Blocking agents are often needed for experiments that use antibodies and other types of proteins. Proteins can non-specifically bind to sticky surfaces such as membranes or plastic tubes and pipettes. If your protein of interest is non-specifically binding to the wrong surface that can lead to erroneous results. Using a blocking agent that is relatively affordable and easy to purchase, such as BSA, prevents your protein of interest, which you may have spent weeks purifying, from engaging in off-target interactions.   

Bovine serum albumin (BSA) is often used as a blocking agent to disrupt non-specific antibody interactions and help reveal specific interacting molecules. BSA has distinct advantages as a blocking agent compared to other options such as dry milk.

We’ll discuss why BSA is used as a blocking agent and touch on some of the experiments that use BSA in this capacity.

 

In this article:

What’s a blocking agent?

Why use BSA as a blocking agent?

Which type of BSA to use as a blocking agent

Experiments using blocking agents

Western blots

ELISA

IHC & IF

References

 

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What’s a blocking agent?

A blocking agent is something that’s used in assays to prevent unwanted and irrelevant background binding. That unwanted background binding could be between your protein of interest and a membrane, a pipette tip, or other plastic surface. Alternatively, the background could arise from weak, non-specific interactions between different molecules.

Including a blocking agent disrupts these non-specific interactions enabling you to focus on the binding events that really matter (Figure 1).

Figure 1. Left, without a blocking agent antibodies will bind non-specifically to the membrane and to random proteins. Right, the blocking agent will mask “sticky” areas, leaving the antibody available to bind to the protein of interest it is supposed to interact with.

 

Why use BSA as a blocking agent?

There are a number of different blocking agents used in molecular biology including BSA, dry milk, detergents, and non-protein polymers such as polyethylene glycol (PEG).

Dry milk is frequently used as a blocking agent, especially for western blots, since it is very affordable and historically it was easier to source; you just walk down to the local grocery store and pick some up.

However, there are some advantages to using a defined blocking agent such as BSA, detergents, or PEG instead of dry milk. We cover some of those advantages below, but an additional one worth pointing out is reproducibility.

Standardized lab reagents like BSA, detergents, and PEG are highly reproducible from lot to lot. Meaning, when you run out of BSA and order some more you’ll get the same blocking effect and lack of background every time.

Dry milk is more variable. It depends on what the cow had to eat, how it slept, the process by which the milk was dried, etc. So, the lot-to-lot variability, meaning the change in performance the next time you pick up a new box of dry milk, is substantial. For example, have you ever paid attention to the recalls for any type of food – this is an alarming sign that something bad changed in how those lots were produced and processed.

 

Which type of BSA to use as a blocking agent

For blocking purposes, you will want to use a version of BSA that is free of proteases and IgG antibodies which have the potential to interfere with your assays. Most of GoldBio’s BSA reagents meet these requirements, with the most cost-efficient versions being:

·         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)

There is an article that reports that blocking with fatty-acid free BSA provides lower background relative to regular BSA (Ma et al, 2020). However, the differences they observe are pretty slight, especially for the assays we’ve discussed in this article (western blotting).

Nevertheless, if you’re experiencing high background in your assay when blocking with one of the BSA products mentioned above, then you may want to use a fatty-acid free version of BSA instead:

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

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

There are some applications of BSA where fatty-acid free versions make a big difference including culturing sensitive cell lines such as CHO, Vera, and MDCK cells.

There are other versions of BSA that would also work for blocking, including sulfhydryl blocked and recombinant human serum albumin. These are specialized versions of BSA that are more expensive. And while they would absolutely work for blocking purposes, you probably want to save these forms for their specific purposes.

 

Experiments using blocking agents

BSA can be used as a blocking agent for a number of different experiments including:

·         Western blotting

·         Enzyme-linked immunosorbent assay (ELISA)

·         Immunohistochemistry (IHC) and Immunofluorescence (IF)

Each of these types of experiments uses antibodies to detect a specific protein of interest, and a blocking agent is useful in preventing the antibodies from binding non-specifically to the membrane or to random proteins. In another article we discuss a similar role for BSA in preventing non-specific protein binding to plastic surfaces, so check that out if you want to learn more.

 

Western blots

Western blots use antibodies to detect a protein of interest on a membrane. First the proteins in the sample being analyzed are separated by size using protein electrophoresis. Then the proteins are transferred from an SDS-PAGE gel onto a membrane. And finally, the protein of interest is detected using an antibody, or combination of antibodies that are either labeled with a fluorophore or linked to an enzyme that triggers a chemiluminescent reaction.

The blocking step comes in here after the proteins have been transferred to the membrane and before adding antibodies to the membrane. The blocking agent will bind to the sticky membrane and to any proteins that may have a weak interaction with the antibody. That way, when the antibody is added to the membrane, it will bind specifically to the protein of interest that it recognizes.

Reconstituted dry milk is often used as the blocking agent for western blots. There are a couple of scenarios where it makes more sense to use BSA instead.

First is if you’re using antibodies that bind to phosphorylated proteins. Casein, a prominent protein in milk, is phosphorylated and so if you block with dry milk then your phospho-specific antibody is going to bind to casein all over the membrane giving you a lot of background.

Second, if you’re using a biotinylated antibody and the streptavidin-biotin interaction for detection, then milk contains trace biotin and glycoproteins that will disrupt this binding event. In this case, you’ll get less signal for your bona fide protein of interest.

So, for cases when you’re using a biotinylated antibody or an antibody that recognizes phosphorylated proteins, BSA is more likely to minimize background instead of milk.

 

ELISA

ELISA is a quantitative assay used to detect specific proteins, hormones or other small molecules, or organisms in a fluid sample. A capture antibody that will recognize the molecule you’re interested in is added first. When the fluid sample is added, the molecule of interest will bind to the capture antibody, and then a detection antibody that also binds to the molecule of interest is added. This antibody is linked to an enzyme, usually horseradish peroxidase (hrp), that generates a chemiluminescent signal, which indicates how much of your molecule of interest is present in the original sample (Figure 2).

Figure 2. In ELISA an antibody captures the molecule of interest from a fluid sample and a detection antibody emits chemiluminescent signal indicating the molecule of interest is present.

A blocking agent is added after the capture antibody has been introduced, and before the fluid sample and detection antibody are added. This blocking agent will coat and prevent your molecule of interest from binding to the plastic plate and prevent other molecules in the sample from binding nonspecifically to the antibody.

BSA is widely used as a blocking agent for ELISAs. The advantages over dry milk are similar. If your detection antibody uses a biotin-streptavidin interaction to link to the chemiluminescent signal, then biotinylated and glycosylated proteins in the milk can disrupt that interaction and make your molecule of interest appear less abundant than it actually is (Hoffman and Jump, 1989).

Additionally, there are reports where using milk as a blocking agent masked the detection of certain targets (Bass et al, 2017). For these reasons, it makes sense to go with BSA, or another standardized lab reagent, so that you can be confident in your results.

In this section I described the “Sandwich” version of ELISAs, which is called that because the molecule of interest is sandwiched between the two antibodies. There are actually multiple types of ELISAs, each with their own considerations. The bottom line is that BSA is a useful blocking agent in each ELISA variation, but if you want to learn more about the different types then this article is a great starting point.

 

IHC & IF

Immunohistochemistry and immunofluorescence are techniques that visualize where a protein is within a tissue section (IHC) or in individual cells (IF). Each of these techniques uses a labeled antibody to bind to the protein of interest, and then advanced microscopy techniques are used to visualize where the labeled antibody, and therefore the protein of interest, are localized within each sample.

For example, researchers used labeled antibodies to identify two proteins that are specifically found on colorectal cancer as a method for identifying these types of cells (Figure 3) (Arul et al, 2014).

Figure 3. Two labeled antibodies bind specifically to a colorectal cancer cell but not to other types of cells like gut microbes.

Blocking is again an important step for IHC and IF. The samples are blocked with BSA, or another blocking agent before adding the detection antibodies to make sure that the antibodies bind specifically to the molecule of interest. Without a blocking agent those researchers would have found that their antibodies bound to everything and were not specific for colorectal cancer cells at all, which illustrates the importance of proper blocking.

The advantages of using BSA as a blocking agent are similar for IHC and IF. If the labeled antibody uses streptavidin-biotin to link to the chemiluminescent enzyme then BSA will be a superior choice instead of dry milk.

 

There are many other uses for BSA, and GoldBio carries different types of BSA to fulfill each niche. So, check out additional articles below to learn more about BSA, and check out the BSA links when you’re ready to order some for your experiments.  

 

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

Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K., & Atherton, P. J. (2017). An overview of technical considerations for Western blotting applications to physiological research. Scandinavian journal of medicine & science in sports, 27(1), 4–25. https://doi.org/10.1111/sms.12702

Hoffman, W. L., & Jump, A. A. (1989). Inhibition of the streptavidin-biotin interaction by milk. Analytical biochemistry, 181(2), 318–320. https://doi.org/10.1016/0003-2697(89)90250-9

Ma, G.J., Ferhan, A.R., Jackman, J.A., Cho, N.J. (2020). Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings. Commun Mater 1, 45 https://doi.org/10.1038/s43246-020-0047-9

 

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Bovine Serum Albumin BSA Simon Currie

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