Q.34 Which of the following method(s) is/are used to estimate protein concentration?
(A) Anthrone
(B) Biuret
(C) Bradford
(D) Lowry
Answer: (B), (C), and (D)
Biuret, Bradford, and Lowry methods measure protein concentration through colorimetric reactions with peptide bonds or amino acids. Anthrone does not target proteins and is unsuitable for this purpose.
Option Analysis
Anthrone (A)
Anthrone reagent reacts with carbohydrates under acidic conditions to form a green-colored complex measured at 630 nm, used for sugar quantification in samples like serum or plant extracts. It hydrolyzes polysaccharides to monosaccharides but shows no specific reaction with proteins, making it incorrect for protein estimation.
Biuret (B)
Biuret reagent, containing Cu²⁺ in alkaline medium, forms a violet complex with peptide bonds in proteins, absorbing at 540 nm; color intensity is proportional to protein concentration. This method suits total protein in serum (6-8 g/dL normal range) but has low sensitivity (above 5 mg/mL).
Bradford (C)
Coomassie Brilliant Blue G-250 dye binds basic/aromatic amino acids under acid conditions, shifting from red to blue (595 nm absorbance) for rapid quantification, ideal for 5-150 μg/mL ranges. It works well for cell lysates but interferes with detergents.
Lowry (D)
Lowry combines Biuret-like Cu²⁺ reaction with Folin-Ciocalteu reagent, reducing it via tyrosine/cysteine to form a blue complex (600-750 nm); highly sensitive (5-150 μg/mL) for diluted samples. It requires multiple steps but excels in enzyme fractions.
Protein concentration estimation methods are essential in biochemistry labs and CSIR NET Life Sciences preparation, enabling accurate quantification for experiments like enzyme assays and gel electrophoresis. Common techniques rely on colorimetric reactions targeting peptide bonds or specific residues, with Biuret, Bradford, and Lowry as gold standards.
Core Principles
These methods use spectrophotometry after protein-dye or protein-metal reactions:
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Biuret: Cu²⁺ chelates peptide bonds (needs ≥2 bonds).
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Bradford: Dye binds arginine, lysine, histidine.
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Lowry: Biuret step plus phosphomolybdate reduction.
UV at 280 nm offers a reagent-free alternative for pure proteins.
| Method | Sensitivity | Wavelength | Time | Interference |
|---|---|---|---|---|
| Biuret | 5-100 mg/mL | 540 nm | 30 min | EDTA, lipids |
| Bradford | 5-150 μg/mL | 595 nm | 5 min | Detergents |
| Lowry | 5-150 μg/mL | 600-750 nm | 40 min | Reducing agents |
CSIR NET Application
For exams, select Biuret, Bradford, Lowry over Anthrone (carbohydrate-specific). Practice standard curves with BSA: plot absorbance vs. concentration for unknowns.
