Aim: To estimate the concentration of proteins by Lowry method from the given sample.
The Blue color developed by the reduction of the Phosphomolybdic-Phosphotungastic components in the Folin-ciocalteau reagent by the amino acids Tyrosine and Tryptophan present in the protein plus the color developed by the biuret reaction of the protein with the color developed by the biuret reaction of the protein with the alkaline cupric tartarate are measured in the Lowry method.
Reagents for Lowry method:
- Lowry Reagent (or) alkaline copper sulfate solution: Mix 50ml 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 tartarate solution (to be prepared fresh)
- Folin-Ciocalteau reagent: This is commercially available and has to be diluted with equal volume of water just before use.
- Standard protein solution: Dissolve 200mg of BSA in 100ml of distilled water in a volumetric flask. (concentration-2mg/ml)
- Working standard: Dilute 10ml of stock standard solution to 100ml of distilled water. (Concentration – 200mg/ml).
Pipette out into a series of tubes 0.2, 0.4, 0.6, 0.8, and 1.0 of the protein solution and make up the total volume to 1ml with addition of water. To each tube 5ml of the alkaline-copper sulfate solution is pipette out, mixed well and allowed to standard at room temperature for 10 to 15 minutes. 0.5ml of the reagent is pipettes out into each tube, mixing rapidly after each addition. The tubes are left as such for 30 minutes and the blue color formed is measured at 700nm ((or) red filter).
Prepare a Blank with 1ml of distilled water, instead of protein solution and with 1ml of unknown solution and proceeds as per standards. Prepare a calibration curve with mg of protein on X-axis and O.D. on Y-axis and determine the amount of protein present in a given unknown sample.
Report: The concentration of protein in the given unknown sample is _____________ mg/ml.