Protocol for protein concentration using the Lowry Protein Assay.
Protein Concentration Determination by the Lowry Prtoein Assay Protocol
1. Complex-forming reagent: Prepare immediately before use by mixing the following stock solutions in the proportion 100:1:1 (by vol), respectively:
Solution A: 2% (w/v) Na2CO3 in distilled water.
Solution B: 1% (w/v) CuSO4·5H2O in distilled water.
Solution C: 2% (w/v) sodium potassium tartrate in distilled water.
2. Prepare 2 N NaOH.
3. Folin reagent (commercially available): Use at 1 N concentration.
4. Standards: Use a stock solution of standard protein (e.g., bovine serum albumin fraction V) containing 2 mg/mL protein in distilled water, stored frozen at –20°C. Prepare standards by diluting the stock solution with distilled water as follows:
Stock solution (µL) 0 2.5 5 12.5 25 50 125 250 500
Water (µL) 500 498 495 488 475 450 375 250 0
Protein conc. (µg/mL) 0 10 20 50 100 200 500 1000 2000
1. To 0.1 mL of sample or standard, add 0.1 mL of 2 N NaOH. Hydrolyze at 100°C for 10 min in a heating block or boiling water bath.
2. Cool the hydrolysate to room temperature and add 1 mL of freshly mixed complex-forming reagent. Let the solution stand at room temperature for 10 min.
3. Add 0.1 mL of Folin reagent, using a vortex mixer, and let the mixture stand at room temperature for 30–60 min (do not exceed 60 min).
4. Read the absorbance at 750 nm if the protein concentration was below 500 µg/mL or at 550 nm if the protein concentration was between 100 and 2000 µg/mL.
5. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations.
Lowry Method References
Based on protocol by Jakob H. Waterborg.
Lowry et al. J. Biol. Chem. 193: 265-275 (1951)
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Lowry Method Protocol