In the classic movie “The Sandlot,” the protagonist Scotty Smalls is asked by his new friends if he would like a s’more. Having never heard of a s’more, Scotty asks how he can have some more when he hasn’t had any yet. Eventually the confusion is resolved by explaining that a s’more is not a request for some more, but rather a treat made out of graham crackers, chocolate, and roasted marshmallows.

In biochemical research, it is frequently useful to know how much protein you have. In order to know if you need some more protein, you need to first know how much you already have. Yet quantifying protein levels in your samples is a little more complicated than recalling if you’ve devoured a s’more yet. And that’s where Bovine Serum Albumin (BSA) comes in.

Bovine Serum Albumin (BSA) is often used as a known standard to calibrate the Bradford, Lowry and BCA assays before quantifying an unknown amount of protein in your samples.

In this article we’ll discuss how BSA is used as a protein standard in common protein concentration measurement assays and briefly touch on the differences between these assays.

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Article Table of Contents:

What is a protein standard?

Why is BSA used as a protein standard?

Assays that use BSA as a protein standard

Bradford Assay

Lowry Assay

BCA Assay

Which type of BSA to use as a protein standard?

References

What is a protein standard?

Think about how you make measurements. You have to use some kind of reference, such as scale or measuring tape to make these measurements.

Standard units of mass measurement use milligrams, grams, kilograms, etc. depending on what scale you’re working at. Similarly, lengths are measured in millimeters, meters, kilometers, etc.

For each of these examples you’re comparing your object to a standard unit of measurement.

Biochemical and molecular biology research often measures proteins in terms of concentration. Certain niche experiments, such as single-molecule experiments deal with proteins in absolute numbers and examine a single protein at a time.

More often, however, we are making bulk measurements about how a group of proteins behave as a collective unit. For these more common experiments it is can be more useful and convenient to know what the concentration of that protein is. The protein concentration is the number of proteins divided by the volume of the solution (Figure 1).

Figure 1. Molar (top) and mass (bottom) versions of protein concentrations.

The concentration we typically use is actually moles of a protein (or other molecule) divided by volume. In this context moles are not the cute little blind animals, but rather it is simply a conversion factor to account for the astronomical numbers in the molecular world. This article is a good starting point to learn more about moles if you’re interested.

That’s the molar concentration, but the other type of concentration that is sometimes used is the mass concentration. Instead of how many proteins you have, this measurement asks what is the mass of the proteins in the solution.

For molar concentrations, the units will be moles per unit volume (mol/L), whereas for mass concentrations the units will be mass by volume (g/L, mg/mL, etc.) (Figure 1).

So, how do you figure out how many proteins you have? Unlike everyday examples, proteins are too small to count by eye. A common way to measure protein concentration involves comparing back to a standard protein to estimate the protein of interest.

This involves making a standard curve with a protein of known concentration, such as BSA, and then comparing your unknown protein concentration to that standard curve.

In other words, by comparing to BSA standards of a known known concentration (Figure 2, blue circles) you will be able to calculate the concentration of your protein of interest in the actual samples that you’re working with (Figure 2, green square).

You know the concentration in your BSA standards because you determined the mass of BSA in your samples and the volume of the buffer, which together dictate the concentration.

Figure 2. BSA standard curve (blue circles). By measuring the absorbance for known concentrations of BSA you can then calculate the concentration of your sample with protein of interest based on its absorbance (green square and dotted lines).

At high enough concentrations of BSA (or any other protein) the signal will start to saturate, so you want to ignore the saturated part and use the linear part of your standard curve to calculate how much protein is in your unknown sample (Figure 2).

Why is BSA used as a protein standard?

BSA is a model protein used for a variety of different purposes in the lab. BSA’s very high solubility (solubility at very high concentrations) and stability are two primary reasons why it is used for so many different purposes.

When it comes to using BSA’s as a protein standard, its high solubility is the main reason it is so useful in this role. In fact, when making a standard curve (Figure 2) it is the saturation of the instrument that limits the upper end of the linear range, rather than BSA’s solubility.

 

Assays that use BSA as a protein standard

Three common assays that measure protein concentration and use a standard protein for calibration are:

·         Bradford

·         Lowry

·         BCA

Each of these assays measures protein concentration in slightly different ways. However, a BSA standard curve, can be used the same way for each assay.

By comparing the signal from your BSA standards of a known concentration, you can calculate the concentration in your experimental samples. Let’s briefly go through the underlying principles for measuring protein concentration in each one.

Bradford Assay

The Bradford assay was developed by Marion Bradford in 1976 and measures the absorbance change of the Coomassie Brilliant Blue dye. By itself, the dye is in its acidic form and appears brown with a maximum absorption at 465 nm wavelength of light.

When Coomassie is bound to protein it converts to its basic form and turns blue with a maximum absorption at 595 nm. That’s why 595 nm is the wavelength at which absorption is measured for Bradford assays (Figure 3).

Figure 3. Free Coomassie dye absorbs light at a smaller wavelength (brown line) relative to dye that is bound to protein (blue line). So, the Bradford assay measures the absorbance of light at wavelength with the maximum absorbance (dotted gray line), which is 595 nm.

Coomassie dye binds to proteins through interactions mainly with the amino acid arginine, though other basic (histidine, lysine) and aromatic amino acids (phenylalanine, tryptophan, tyrosine) also contribute to lesser extents (Compton & Jones, 1985). If you want to review the types of amino acids and their properties, this article is a great place to start.    

 

Lowry Assay

The Lowry method for determining protein concentration was developed in 1951 by Oliver Lowry. This is a multistep reaction. First, the backbone peptide bonds in the protein react with copper ions to form a copper-peptide complex. Together the copper-peptide complex and side chains from the amino acids tyrosine, tryptophan, and cysteine reduce Folin-Ciocalteu reagent, which is phosphomolybdic or phosphotungstic acid. This final reaction creates a blue-green product which absorbs light between 650 and 750 nm.

 

BCA Assay

The bicinchoninic acid (BCA) assay is pretty similar to Lowry’s method for quantifying proteins. The first step is the same in that the peptide bonds from the protein bind to copper. Then, BCA is added to the reaction and when it binds to copper it generates a purple color that strongly absorbs light at 562 nm.

 

As you can tell, each of these assays use different characteristics to measure protein concentration. One thing to keep in mind is that the closer your protein of interest is to the standard protein in terms of each assay’s characteristics the better your estimation of protein concentration will be.

Usually, BSA is similar enough to most proteins that it is a good choice to use as a standard in each of these assays. That is, the abundance and patterning of amino acids that drive the signal in Bradford, Lowry, and BCA assays is close enough between BSA and your protein of interest to use BSA standards to calculate your protein’s concentration. But, if for example you were working with an outlier protein – say a small peptide devoid of arginine residues – then BSA might not be the most representative protein standard that you could use for Bradford. Instead, a small protein or peptide that is closer in size and amino acid composition to your peptide of interest would be a better choice. 

 

Which type of BSA to use as a protein standard?

Most versions of BSA should work just fine as a protein standard. Below we list three affordable versions of BSA that would be great for this purpose:

·         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)

 

Now that you’re ready to make BSA standard curves in Bradford, Lowry, or BCA assays, check out our selection of affordable and reliable BSA in the links below. Also, remember that BSA is not just a protein standard, but also has many other uses in the lab. Check out the articles below to learn more about all of the ways that BSA can help support your research.

 

References

Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3

Compton, S. J., & Jones, C. G. (1985). Mechanism of dye response and interference in the Bradford protein assay. Analytical biochemistry, 151(2), 369–374. https://doi.org/10.1016/0003-2697(85)90190-3

LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of biological chemistry, 193(1), 265–275.

Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., & Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical biochemistry, 150(1), 76–85. https://doi.org/10.1016/0003-2697(85)90442-7

 

 

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