Question

Discussion Questions Your discussion question for this week's lab exercise deals with the three types of protein quantification methods discussed in this section. Though each method, Biuret reaction, BCA, and Bradford assays was briefly described, for your discussion question, compare and contrast in chemical detail the three methods. Explain if any the pros and cons of each method, and why a protein scientist would prefer one method to another. Finally, suggest and describe other types of protein quantification methods not mentioned here.

Answer 1

The biuret test (Piotrowski's test) is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colored coordination complexes in an alkalinesolution.Several variants on the test have been developed, such as the BCA test and the Modified Lowry test.

The biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.

Despite its name, the reagent does not in fact contain biuret ((H₂N-CO-)₂NH). The test is named so because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.

In this assay, the copper(II) binds with nitrogens present in the peptides of proteins. In a secondary reaction, the copper(II) is reduced to copper(I). Buffers, such as Tris and ammonia interfere with this assay, therefore rendering this assay inappropriate for protein samples purified from ammonium sulfate precipitation. Due to its insensitivity and little interference by free amino acids, this assay is most useful for whole tissue samples and other sources with high protein concentration.

Bradford

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.

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.

What to use?

Bradford vs BCA?

BCA assay is less sensitive to detergents thats the most common reason why you use it. So if you are using a lysisbuffer (without reducing agents) Bca might be the beter one.

Detergents bind the Coomassie dye and therefore artificially contribute to signal.Reducing agents react with the Cu2+ ion in the BCA mixture and therefore artificially contribute to signal.

Therefore:

Use the BCA assay if your samples contain non-ionic detergents (Triton, NP-40, Tween, etc.). But use the Bradford assay if your samples contain reducing agents (DTT, BME, TCEP, etc.).

CONCLUSION :-

As the conclusion, the Lowry technique seems to be the best method in determining the protein

concentration of hen egg, because Biuret assay is not much sensitive and Bradford can be inhibited by

the presence of many compounds.