What could possibly be easier and sooner than measuring the quantity of protein in your pattern? Properly, don’t get too assured – even essentially the most humble protein quantification assay makes use of some subtle chemistry that may journey you up (particularly should you’re working with detergents!). That will help you select the appropriate methodology to “simply” measure your protein, I wrote this fast overview masking the 5 predominant protein quantification strategies – how they work, once they work, and once they don’t work.
Bicinchoninic Acid (BCA)
This colorimetric, two-step assay was initially developed in 1985 – making it a child in contrast with the 64-year-old Lowry assay! Just like the Lowry assay, step one right here is to complicated the protein with copper ions. Within the second step, this protein-bound copper chelates BCA to provide an intense purple colour. Conveniently, the purple’s depth is linear with the quantity of protein. Much less conveniently, every pattern’s depth have to be in comparison with a typical curve as a result of (in contrast to the Folin-Lowry methodology) this assay doesn’t have a set endpoint, due to the extreme quantity of its reagents in contrast together with your pattern.
Whereas slower than the Bradford, the BCA assay is a superb choice in case your protein samples include > 5% detergents. It additionally has a extra uniform response to totally different proteins than the Bradford assay, though it’s nonetheless strongly influenced by the presence of tyrosine, tryptophan, and cysteine amino acids. Nevertheless, as a result of it depends on copper for that first response, chemical compounds which work together with copper (akin to ammonia) can even intrude with the BCA assay.
Bradford
There are good causes that the paper first describing this colorimetric methodology has been cited hundreds of instances! The Bradford methodology is elegantly easy: negatively-charged Coomassie sensible blue dye binds to positively-charged proteins. When the dye is in answer, it’s purple and absorbs at 465 nm – however when it binds to fundamental amino acids within the protein, it turns into blue and absorbs at 595 nm. The absorption in your pattern can then be in comparison with a typical curve.
The Bradford response is quick, straightforward, and secure for as much as an hour. Nevertheless, it typically can solely detect proteins bigger than 3 kDa. In contrast to the BCA, it’s delicate to detergents like SDS and Triton X-100.
Folin-Lowry
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 pals) to provide an intense, blue-green colour which absorbs at 650–750 nm. In contrast to BCA, that is an endpoint assay with a secure outcome, that means which you can estimate the quantity of protein from one assay by evaluating it with a earlier normal curve!
Sadly, this assay isn’t suitable with a lot of widespread chemical compounds: EDTA, Tris, carbohydrates, lowering brokers (e.g., DTT, 2-mercaptoethanol), and potassium and magnesium ions are all incompatible.
Kjeldahl
This 132-year-old methodology measures the nitrogen in a protein pattern after it’s been transformed to ammonia via a sequence of terrifying steps involving heated sulfuric acid, steam distillation, and back-titration with sodium hydroxide. In spite of everything that work, you weigh out your purified nitrogen and – by assuming that your unique protein pattern was 16% nitrogen – back-calculate the whole quantity of protein. Whew!
Tedious and time-consuming, the Kjeldahl methodology requires at the least 1 gram of pattern, making it extremely impractical for many molecular biologists!
Ultraviolet Absorption – “protein quantification”
Easy however usually unreliable, this methodology estimates the quantity of protein by measuring the attribute absorption of tyrosine and tryptophan at 280 nm. Nevertheless, each protein has a distinct quantity of those amino acids! And as if that didn’t make this method unreliable sufficient, a lot of different molecules intrude with this method. Alcohols, sure buffer ions, and nucleic acids all take up at 280 nm, thereby making this methodology non-specific for protein if any of those different molecules are current.
With all of the totally different protein assays on the market, it’s necessary to know the fundamentals! To be taught extra about these assays and others, go to the references beneath, or try our Protein Evaluation, Detection & Assay channel.
References:
“Chemistry of Protein Assays.” Life Applied sciences. 2015.
Robyt JF and White BJ. Biochemical Methods: Idea and Observe. Chapter 7: Strategies for Figuring out Organic Molecules. 1987. Waveland Press, Inc.
“protein quantification”