What could be simpler and faster than measuring the amount of protein in your sample? Well, don’t get too confident – even the most humble protein quantification assay uses some sophisticated chemistry that can trip you up (especially if you’re working with detergents!). To help you choose the right method to “simply” measure your protein, I wrote this quick overview covering the five main protein quantification methods – how they work, when they work, and when they don’t work.
Bicinchoninic Acid (BCA)
This colorimetric, two-step assay was originally developed in 1985 – making it a baby compared with the 64-year-old Lowry assay! Like the Lowry assay, the first step here is to complex the protein with copper ions. In the second step, this protein-bound copper chelates BCA to give an intense purple color. Conveniently, the purple’s intensity is linear with the amount of protein. Less conveniently, each sample’s intensity must be compared to a standard curve because (unlike the Folin-Lowry method) this assay doesn’t have a set endpoint, thanks to the excessive amount of its reagents compared with your sample.
While slower than the Bradford, 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 which interact with copper (such as ammonia) can also interfere with the BCA assay.
There are good reasons that the paper first describing this colorimetric method has been cited thousands of times! The Bradford method is elegantly simple: negatively-charged Coomassie brilliant blue dye binds to positively-charged proteins. When the dye is in solution, it’s red and absorbs at 465 nm – but when it binds to basic amino acids in the protein, it becomes blue and absorbs at 595 nm. The absorption in your sample can then be compared to a standard curve.
The Bradford reaction is fast, easy, and stable for up to an hour. However, it generally can only detect proteins larger than 3 kDa. Unlike the BCA, it’s sensitive to detergents like SDS and Triton X-100.
This good ol’ colorimetric assay works in two steps: 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. Unlike BCA, 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!
Unfortunately, this assay isn’t compatible with lots of common chemicals: EDTA, Tris, carbohydrates, reducing agents (e.g., DTT, 2-mercaptoethanol), and potassium and magnesium ions are all incompatible.
This 132-year-old method 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. 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!
Tedious and time-consuming, the Kjeldahl method requires at least 1 gram of sample, making it highly impractical for most molecular biologists!
Simple but often unreliable, this method estimates the amount of protein by measuring the characteristic absorption of tyrosine and tryptophan at 280 nm. However, every protein has a different amount of these amino acids! And as if that didn’t make this approach unreliable enough, lots of other molecules interfere with this approach. Alcohols, certain buffer ions, and nucleic acids all absorb at 280 nm, thereby making this method non-specific for protein if any of these other molecules are present.
With all the different protein assays out there, it’s important to understand the basics! To learn more about these assays and others, go to the references below, or check out our Protein Analysis, Detection & Assay channel.
“Chemistry of Protein Assays.” Life Technologies. 2015.
Robyt JF and White BJ. Biochemical Techniques: Theory and Practice. Chapter 7: Methods for Determining Biological Molecules. 1987. Waveland Press, Inc.