Lowry Protein Estimation Method Protocol

Lowry Protein Estimation Protocol

Aim

To estimate the protein using Lowry’s method.

Principle

The –CO-NH– bond (peptide bond) in the polypeptide chain reacts with copper sulphate in an alkaline medium to produce a blue-colored complex. Additionally, tyrosine and tryptophan residues of the protein reduce the phosphomolybdate and phosphotungstate components of the Folin-Ciocalteau reagent, yielding bluish products that enhance the sensitivity of this method.

Reagents Required

• Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide.
• Reagent B: 0.5% copper sulphate (CuSO₄·5H₂O) in 1% potassium sodium tartarate. Prepare fresh by mixing stock solutions.
• Alkaline Copper Solution (Reagent C): Mix 50 mL of Reagent A and 1 mL of Reagent B prior to use.
• Diluted Folin’s Reagent (Reagent D): Dilute Folin-Ciocalteau reagent with an equal volume of 0.1 N NaOH.
• Standard: Dissolve 50 mg BSA in 50 mL of distilled water in a volumetric flask. Take 10 mL of this stock standard and dilute to 50 mL in another flask for the working standard solution. One mL of this solution contains 200 µg protein.

Apparatus and Glassware Required

Test tubes, pipettes, colorimeter, etc.

Procedure

1. Pipette out 0.2, 0.4, 0.6, 0.8, and 1 mL of working standard into a series of labeled test tubes.
2. Pipette out 1 mL of the sample into another test tube.
3. Make up the volume to 1 mL in all test tubes. A tube with 1 mL of distilled water serves as the blank.
4. Now add 5 mL of Reagent C to all test tubes, including the tubes labeled 'blank' and 'unknown'.
5. Mix the contents of the tubes by vortexing or shaking, and allow them to stand for 10 minutes.
6. Then, add 0.5 mL of Reagent D rapidly with immediate thorough mixing, and incubate at room temperature in the dark for 30 minutes.
7. Now record the absorbance at 660 nm against the blank.
8. Plot the standard curve by taking the concentration of protein along the X-axis and absorbance at 660 nm along the Y-axis.
9. From this standard curve, calculate the concentration of protein in the given sample.

Result

The given unknown sample contains ______ µg protein/mL.