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Top 5 Protein Quantification Assays

Posted in: Protein Expression and Analysis
Colorful wells in 96-well plate to represent colorimetric protein quantification assays

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Accurate protein quantification is key when working with proteins. But there are so many methods out there, how do you know which one is right for you?

In this article, we’re going to discuss five major protein quantification techniques—how they work, when they work, and when they don’t work.

Why is Accurate Protein Quantification Important?

Accurate protein quantification is critical if, for example, you’re trying to determine a binding constant, measure enzyme kinetics, or if you’re preparing samples for a western blot. Even if you’re doing something more qualitative, having a good idea of how much protein you have will enable you to compare results from one experiment to the next and from one protein to the next.

There are several ways to measure protein concentration, and each of them has its own advantages and disadvantages. It can be difficult to decide on the best method for your protein, especially considering that even the most humble protein quantification assay uses some pretty sophisticated chemistry that can trip you up (particularly if you’re working with detergents!).

UV-Vis Absorbance at 280 nm

Simple but often unreliable, this protein quantification method estimates the amount of protein by measuring the characteristic absorption of the aromatic residues, tyrosine, and tryptophan, at 280 nm on a UV-Vis spectrometer.

Once you know the absorbance of your protein at 280 nm (A280), as well as its extinction coefficient, you can use the Beer–Lambert law to calculate protein concentration:

A = ε l c


A = absorbance at 280 nm.

ε = the molar extinction coefficient.

l = path length of the spectrometer.

c = molar concentration of protein.

Advantages of Measuring Protein Concentration using Absorbance at 280 nm

This is a quick method and doesn’t require any special reagents, except for the guanidinium, which you may have on hand anyway.

If you’re working with purified protein samples and if you measure the complete UV-vis spectrum of your protein sample rather than just the A280, you can also see if there are any soluble aggregates in your sample by looking for absorbance at 230 nm. (1)

Disadvantages of Measuring Protein Concentration using Absorbance at 280 nm

Every protein has a different number of tyrosine and tryptophan residues and, annoyingly, you may not know the experimental extinction of your protein. These factors alone make this approach unreliable.

To make matters worse, lots of other molecules interfere with this method of protein quantification. Alcohols, certain buffer ions, and nucleic acids all absorb at 280 nm, thereby making this measurement non-specific for protein if any of these other molecules are present.

If you use DTT in your protein preps and use A280 to measure protein concentration—be careful! DTT oxidizes over time leaving a product that also absorbs at 280 nm. This doesn’t mean that UV-Vis is incompatible with DTT, it just means that you should use an exact DTT-containing buffer match to measure protein concentration more accurately.

This can be difficult to do especially if you freeze protein and use it for weeks/months but you no longer have the exact buffer to blank against. In which case, one of the following techniques might be more useful for you.

The Bradford Assay

There are good reasons that the paper first describing the Bradford Assay has been cited thousands of times! (2) The Bradford assay is an elegantly simple colorimetric assay for protein quantification.

It’s based on the interaction between Coomassie brilliant blue (you know, the stuff you stain your SDS-PAGE gels with) and the arginine and aromatic residues in your protein. When the dye binds to these residues, its maximum absorption shifts from 470 nm to 595 nm. 

In general, you measure the absorbance of a series of known concentrations of a standard protein [e.g., bovine serum albumin (BSA) or bovine γ-globulin (BGG)] to create a standard curve. Unlike the Folin-Lowry method, the Bradford assay doesn’t have a set endpoint, so you have to use this standard curve to calculate protein concentration based on its absorbance.

Advantages of the Bradford Assay

Bradford protein quantification is quick, easy, stable for up to an hour, and isn’t affected by the presence of reducing agents in your buffer, like DTT or my favorite smelly lab chemical, β-mercaptoethanol.

Disadvantages of the Bradford Assay

However, if you’ve your standard protein dilutions aren’t accurate, then your calculated protein concentration will be off. Importantly, if your protein doesn’t react to the dye with similar chemistry as your standard protein, your concentrations can also be incorrect.

Unfortunately, basic conditions and detergents, such as SDS, can interfere with the dye’s ability to bind to proteins. However, there are detergent-compatible Bradford reagents.

In addition, like the UV-vis A280 technique, the Bradford assay depends on the sequence of your protein. If your protein doesn’t contain a decent number of arginine and/or aromatic residues, then the dye will not bind to the protein as efficiently, resulting in an underestimation of your protein concentration.

The Bicinchoninic Acid (BCA) Assay

Originally developed in 1985, the BCA assay is another colorimetric assay. (3) This two-step assay first makes use of the biuret reaction, in which the protein backbone chelates Cu2+ ions and reduces them to Cu+ ions. In the second step, the Cu+ ions react with BCA to form a purple-colored product that absorbs at 562 nm. 

Conveniently, the color intensity is proportional to the amount of protein and, like the Bradford assay, each sample’s intensity must also be compared to a standard curve based on a series of known protein standards.

Advantages of the BCA Assay

Because the peptide backbone is involved in the reaction, the BCA assay is less affected by differences in the amino acid composition. However, the reaction is influenced by cysteine, tyrosine, and tryptophan residues. In addition, BCA reagents are not sensitive to detergents and denaturants, so it’s okay to have those in your buffer.

This assay can easily be done in a 96-well plate for improved convenience and to allow for high throughput protein quantification.

Disadvantages of the BCA Assay

The presence of reducing agents in your buffer can interfere with the assay, but there are reducing agent-compatible kits available.

The reaction takes some time to proceed. Usually, the samples are incubated at 37°C for 15–30 min. Just enough time to grab a coffee. Also, as in the Bradford assay, you determine your protein concentration by creating a standard curve from a known, standard protein. So again, the same pitfalls apply.

While slower than the Bradford assay, the BCA assay is a great option if your protein samples contain >5% detergents. It also has a more uniform response to different proteins than the Bradford assay, although it’s still strongly influenced by the presence of tyrosine, tryptophan, and cysteine amino acids.

However, because it relies on copper for that first reaction, chemicals that interact with copper (such as ammonia) can also interfere with the BCA assay.

The Folin–Lowry Assay

Similar to the BCA assay, this good ol’ colorimetric assay also involves two steps, one of which also includes the biuret reaction. First, it complexes copper with the nitrogen in your protein; second, the complexed tyrosine and tryptophan react with Folin–Ciocalteu phenol reagent (“phosphomolybdotungstate” to its friends) to give an intense, blue-green color which absorbs at 650–750 nm. This blue color intensifies during the 30-minute incubation at room temperature. (4)

Advantages of the Folin–Lowry Assay

One advantage of the Folin–Lowry assay lies in the flexibility to measure at any wavelength between 650 nm and 750 nm with little loss of color intensity. Despite this, it’s best to measure at 750 nm because few other substances absorb at this wavelength. However, it’s important to remember that, unlike the BCA assay, a Folin–Lowry standard curve is nonlinear.

Another advantage is that this is an endpoint assay with a stable result, meaning that you can estimate the amount of protein from one assay by comparing it with a previous standard curve!

Disadvantages of the Folin–Lowry Assay

Unfortunately, this assay isn’t compatible with lots of common chemicals: EDTA, Tris, carbohydrates, reducing agents (e.g., DTT, β-mercaptoethanol), as well as potassium and magnesium ions are all incompatible.

Annoyingly, the Folin phenol reagent must be added to each sample precisely at the end of the 10-minute incubation period. If you have lots of samples to measure this can limit the number of samples you can assay in one run and you may need to practice to get consistent results. Not great for those of us who like to go big or go home!

The Kjeldahl Method

The Kjeldahl method is a 132-year-old method that measures the nitrogen in a protein sample after it’s been converted to ammonia through a series of terrifying steps involving heated sulfuric acid, steam distillation, and back-titration with sodium hydroxide. (4, 5) After all that work, you weigh out your purified nitrogen and—by assuming that your original protein sample was 16% nitrogen—back-calculate the total amount of protein. Whew!

Advantages of the Kjeldahl Method

The strength of this method lies in its precision and reproducibility. It’s used to assay protein content in food, soils, wastewater, and fertilizers.

Disadvantages of the Kjeldahl Method

Tedious and time-consuming, the Kjeldahl method requires at least 1 gram of sample, making it highly impractical for most molecular biologists, especially if like me, you struggle to produce microgram amounts of your protein of interest!

In addition, this method measures non-protein nitrogen as well as the nitrogen in proteins and it doesn’t give an accurate measure of true protein content.

Protein Quantification Assays Summarized

Table 1 summarizes the key advantages and disadvantages of each protein quantification method.

Table 1. Top 5 protein quantification assays




Absorbance at 280 nm

Quick, easy, and doesn't require many special reagents.

Relies on the presence of tryptophan and tyrosine residues in protein sequence.

Exact extinction coefficient of protein may be unknown. 

Alcohols, some buffer ions, and nucleic acids interfere with assay. 

Need to be careful if using DTT in protein storage buffer.


Simple and stable assay.

Not affected by reducing agents.

Detergent compatible assay kits are available.

Requires standard protein dilutions. If these dilutions are inaccurate, then the calculated protein concenration will be incorrect.

Detergents interfere with assay results.

Need a decent number of arginine and/or aromatic residues for efficient dye binding.


Less affected by amino acid composition.

Not influenced by  detergents and denaturants.

High throughput.

Detergent compatible assay kits are available.

Gives more uniform response than the Bradford assay.

Requires presence of cysteine, tyrosine, and tryptophan residues.

Incompatible with reducing agents.

Requires 15-30 min incubation time.

Requires standard protein dilutions. If these dilutions are inaccurate, then the calculated protein concenration will be incorrect.

Incompatible with copper-interacting reagents (e.g. ammonia).


Measurable at any wavelength between 650 nm and 750 nm.

Is an end-point assay with a stable signal.

Doesn't require use of a standard curve.

Incompatible with many chemicals (e.g. EDTA, Tris, carbohydrates, DTT, β-mercaptoethanol, potassium and magnesium ions).

Assay components must be added at specific time points, which may require some practice to get reproducible and reliable results.


Precise and reproducible.

Tedious and time-consuming.

Requires gram amounts of protein.

Doesn't give accurate measure of true protein content.

With all the different protein quantification assays out there, it’s important to understand the basics! To learn more about these protein quantification assays and others check out our Protein Analysis, Detection & Assay channel.

Did we miss your favorite protein quantification method? Leave us a comment below.

Want do you want a stunning, colorful poster that summarizes all the critical fluorescent protein properties like absorption and emission spectra, relative brightness, and quantum yield? Download Bitesize Bio’s ultimate guide to fluorescent proteins poster and stick it up in your lab!

Originally published May 27, 2015. Updated and republished June 2022.


  1. Liu PF, et al. (2009) Revisiting absorbance at 230nm as a protein unfolding probe. Anal Biochem. 389(2):165-70.
  2. Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72:248-54.
  3. Smith PK, et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem. 150(1):76-85.
  4. “Chemistry of Protein Assays.” Life Technologies 2015.
  5. Robyt JF and White BJ. Biochemical Techniques: Theory and Practice. Chapter 7: Methods for Determining Biological Molecules. 1987. Waveland Press, Inc.
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