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What are the standard chemicals used in lab for protein estimation?
Wiki User
∙ 7y ago
There are several methods for protein estimation, these are the most common:
- Near UV Absorbance (280 nm). Quantitation of the amount of protein in a solution using a simple spectrophotometer. UV detection focuses on Tyr and Trp content, and to a very small extent on the amount of Phe and disulfide bonds.
- Far UV Absorbance (205 nm). Detects various side chains, including those of Trp, Phe, Tyr, His, Cys, Met, and Arg.
These two methods uses: 0.1 M K2SO4 (pH 7.0), 5 mM KPO4 buffer (pH 7.0), Non -ionic detergent (e.g., 0.01% Brijn 35), and Guanidinium-HCl.
- The Lowry Method. This method is based on both the Biuret reaction, in wich the peptide bonds of proteins react with cooper under alkaline conditions to produce Cu+, which reacts with the Folin reagent, and the Folin-Ciocalteau reaction, where phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the cooper-catalyzed oxidation of aromatic amino acids.
This methods requires: Complex-forming reagent (composed by the Solution A: 2% (w/v) Na2CO3 in dH2O; Solution B: 1% (w/v) CuSO4 5H2O in dH2O; Solution C: 2% (w/v) sodium potassium tartrate in dH2O); 2 N NaOH; Folin Reagent (commercially available); and protein standards (bovine serum albumin fraction V is widely used).
- The Bicinchoninic Acid (BCA) Assay. This method is similar to Lowry method, since it also depends on the conversion of Cu2+ to Cu+ under alkaline conditions. The Cu+ is then detected by reaction with BCA.
This methods requires: Reagent A (0.1 g sodium bicinchoninate, 2.0 g Na2CO3 H2O, 0.16 g sodium tartrate dihydrate, 0.4 g NaOH, 0.95 g NaHCO3, made up to 100 mL. Adjust pH to 11.25 with NaHCO3 or NaOH, if necessary; Reagent B (0.4 g CuSo4 5H2O in 10 mL of water). The Standard Working Reagent (SWR) is made by mixing 100 vol of A + 2 vol of B.
- The Bradford Assay. This method relies on the binding of the dye Coomassie Blue G250 to protein.
This assays requires a solution of 100 mg Coomassie Blue G250 in 50 mL of 95% ethanol, mixed with 100 mL of 85% phosphoric acid and completed to 1 L with distilled water.
These are the most widely used assays for protein estimation.
Wiki User
∙ 7y ago
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