Aim: To estimate the concentration of proteins by the Lowry method from the given sample.

Table of Contents

Principle

The blue color developed in the Lowry method results from the reduction of phosphomolybdic-phosphotungstic components in the Folin-Ciocalteau reagent. This reduction occurs due to the presence of the amino acids tyrosine and tryptophan in the protein. Additionally, the color developed by the biuret reaction of the protein with alkaline cupric tartrate is measured by the Lowry method.

Reagents

Lowry Reagent (or) alkaline copper sulfate solution: Mix 50 ml of solution A with 1 ml of solution B just prior to use.
- Solution A: 2% sodium carbonate in 0.1N NaOH.
- Solution B: 0.5% copper sulfate solution in 1% sodium potassium tartrate solution (to be prepared fresh).

Folin-Ciocalteau reagent: commercially available, diluted with an equal volume of water just before use.
Standard protein solution: Dissolve 200mg of BSA in 100 ml of distilled water in a volumetric flask. (concentration: 2 mg/mL).
Working standard: Dilute 10 ml of the stock standard solution with 100 ml of distilled water. (concentration: 200 mg/mL).

Procedure

Pipette out 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the protein solution into a series of tubes. Make up the total volume to 1 ml with the addition of water.
To each tube, pipette out 5 ml of the alkaline-copper sulfate solution, mix well, and allow it to stand at room temperature for 10 to 15 minutes.
Pipette out 0.5 ml of the Folin-Ciocalteau reagent into each tube, mixing rapidly after each addition.
Leave the tubes for 30 minutes, and measure the blue color formed at 700nm (or a red filter).
Prepare a blank with 1 ml of distilled water instead of the protein solution and with 1 ml of the unknown solution, and proceed as per standards.
Prepare a calibration curve with mg of protein on the X-axis and O.D. on the Y-axis. Determine the amount of protein present in the given unknown sample.

Report

The concentration of protein in the given unknown sample is _____________ mg/ml.

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