Have you ever spent ages correctly quantifying your protein samples, casting the perfect gel, efficiently transferred, probed and eventually come to the visualization stage only to be presented with a loading control like this:
Factors to consider when choosing the right protein assay
Before you throw your blot out with the bathwater and repeat the experiment, you should consider how your choice of protein assay can interact with your samples. We’re here to help you detect the presence of protein.
The two most common methods for the colorimetric detection and quantitation of total protein are the protein-copper ion-based and protein-dye binding assays. We’ll cover both types and more in this blog post.
Four major methods of determining protein concentrations
Most modern protein assays are based on two colorimetric assays. The first relies on the interaction of proteins with copper and the subsequent reduction of these copper ions (Biuret and its derivations BCA and Lowry). The second on direct alterations to Coomassie Blue dye due to its interaction with proteins in solution (Bradford). Both the protein sample’s nature and presence of non-protein agents can significantly affect the results of your experiment.
Keep in mind most colorimetric assays require at least 0.5µg proteins in order to produce reliable estimations. You will need proper instrumentation to measure the absorbance. Using an absorbance microplate reader with a highly sensitive and reactive read speed and high accuracy rate will allow you to quickly get your results. We recommend using the Ao Absorbance Microplate Reader, which a read speed of <6 seconds, and an accuracy of >0.005 +/- 1% (0-3) OD. Using this reader, you can comfortably and quickly run any assay using an intuitive 7” touchscreen, without the need for a separate computer.
Table 1. How to choose a protein assay
|Choose this protein assay
|You see odd looking results even though you're sure your quantification is accurate and your loading control is correct
|Look at your protein assay again
|Your extraction buffers contain reducing agents such as DTT or BME
|Bradford protein assay
|Your samples contain detergents
|Either BCA or Lowry assay
|You're working with high concentration samples
|Biuret assay. Keep in mind it is often preferable to simply dilute high concentration samples down.
Types of Protein-copper Assays
Protein-copper assays based are divided into two groups: 1) assays that detect reduced cuprous ions (Cu+) and 2) assays that detect unbound cupric (Cu2+) ions. Bicinchoninic acid (BCA) may be used to detect cuprous ions.
The biuret test (also called Piotrowski’s test) was first described in 1833, although its usage as a protein assay came much later. Given its relative insensitivity (how many of us are using samples in the range of 5 to 150 mg/mL) and lack of reactivity with single amino acids or small peptides, the biuret assay is ideal for high protein samples, such as tissue.
At its most simple an aqueous protein sample is treated with as strong base (typically sodium hydroxide NaOH) and a copper(II) sulfate solution. When proteins are present the copper(II) binds with nitrogens present in the protein sample. These liberated copper(II) molecules can now be reduced which in turn changes the color of the solution from a pale blue into a nice shade of purple as seen below. As the amount of reduced copper ions are correlated with the amount of protein present in the sample, and in conjunction with a standard curve, it is then possible to accurately quantify proteins within the range of 5 to 150 mg/mL.
There are two derivations of the biuret assay which be used to dramatically increase assay sensitivity: the BCA and Lowry assays.
Bicinchoninic acid (BCA) Assay
The BCA assay is a high-throughput colorimetric assay that gives accurate quantitation. In the BCA assay the copper ions generated with through interaction with proteins react with BCA, producing a strong violet or blue-ish color. The difference between biuret assays and BCA assays, however, is the formed BCA/copper complex absorbs light much more strongly than those generated by the biuret assay. For this reason alone, the assay’s sensitivity is greatly increased.
With proper sample preparation and accurate standard curves, it is possible to assay samples accurately within the 0.001 to 2 mg/mL protein concentration range.
It’s important to note the color density is affected by the presence of asparginine, tyrosine, cysteine, tryptophan, and histidine. Avoid EDTA (>10 mM) and other strong chelating agents with BCA assays. The color produced (absorbance) can be read at 650nm to 750nm and is proportional to the amount of peptide bonds. BCA assays are incompatible with reducing agents and copper-interacting reagents like ammonia.
The Lowry assay is an earlier derivation developed by Oliver Lowry in the 1940’s. In a similar fashion to the BCA assay, the Lowry assay relies on the reaction of copper ions, produced from the interaction of copper(II) sulfate with proteins present in the sample, with a further reagent. In this case Folin–Ciocalteu reagent with the sample becoming blue in color when higher levels of protein are present. This assay can be a tad tricky, as it’ll take practice to add certain components need to be added at specific time points. You will be able to measure the Lowry method between 650 nm and 750 nm without loosing color intensity.
The Lowry method is sensitive in the range 5 to 150 μg protein per assay. It is often noted as more consistent than the Bradford assay. Because of its sensitivity to low molecular weight peptides and proteins, the Lowry method is able to more accurately evaluate the increase of purity during fractionation.
However, the three protein-copper assays described above are sensitive to reducing agents such as dithiothreitol (DTT) or beta-mercaptoethanol (BME), which are often used in sample preparations. These reducing agents can inflate protein concentration values by increasing the reduction of copper ions.
Types of Protein-Dye Assays
Protein-dye assays are not sensitive to reducing agents; however, they are sensitive to detergents, such as Triton, Tween or sodium dodecyl sulfate (SDS).
The Bradford assay is fast to perform and gives accurate quantitation. It uses the same amount of protein as the Lowry assay. The Bradford assay relies on an absorption shift by Coomassie Blue when it binds to proteins present in a solution. Under acidic conditions unbound Coomassie will present its typical blue-green color with a maximal absorbance of 465 nm.
So… Which protein assay to chose?
- If you see odd looking results even though you’re sure your quantification is accurate and your loading control is correct: consider looking at your protein assay again
- If your extraction buffers contain reducing agents such as DTT or BME: consider the Bradford protein assay
- If your samples contain detergents: BCA or Lowry assay may be preferred.
- If you’re working with high concentration samples: the biuret assay may be used. Keep in mind that it is often preferable to simply dilute high concentration samples down.
However, under the same conditions, Coomassie readily binds non-covalently with the carboxyl groups of proteins which induces a conformational shift allowing for a further ionic bond to form between the protein and the Coomassie dye. This binding alters the absorbance profile of the dye and changes the color from brown to blue, which can be measured at 595 nm. By measuring the shift from 465 nm to 595 nm and in conjunction with an appropriate standard curve, accurate protein quantification is possible.
The main limitation of Bradford assays is that it is not compatible with most detergents. Another major drawback of the Bradford protein assay is that it does not work if the sample is basic, or surfactants or detergents are in the sample.
Still unsure about which protein assay to choose? We’re here for you! Fill out the form on this page to ask a question.