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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
LOWRY ASSAY FOR PROTEIN DETERMINATION
Lowry Assay for Protein Determination
Contributor: The Laboratory of Andy Golden at the National Cancer Institute Overview
This protocol describes the Lowry method for protein concentration determination. Procedure
1. In triplicate set up a standard curve containing between 0 and 50 μg of BSA Stock in a final volume of 100 μl. (See Hint #1)
2. In triplicate add 100 μl of unknown sample.
3. To each tube add 1 ml of Lowry Solution, vortex for 5 sec and incubate tube at room temperature for 10 min.
4. To each tube add 100 μl of Phenol Mix, vortex for 5 sec and incubate at room temperature for 20 min.
5. Read each tube at 750 nm (See Hint #2)
BSA Stock Solution 1 mg/ml Bovine Serum Albumin Fraction V Phenol Mix 50% Folins Reagent diluted in ddH2O Lowry Solution 200 μl of Lowry A
10 ml of Lowry B
Make up this solution just before performing the assay.
Lowry B 0.1 N NaOH
2% Na2CO3 anhydrous
Lowry A 2% (w/v) Sodium Tartrate
1% (w/v) Copper Sulfate Pentahydrate
BioReagents and Chemicals
Sodium Carbonate Anhydrous
Copper Sulfate Pentahydrate
Bovine Serum Albumin
1. In triplicate make a five point standard containing 0, 10, 20, 35 and 50 μg of BSA Stock, bringing the final volume up to 100 μl.
2. The reading goes up about 5% one hour after the scheduled waiting time of 20 min. Also there is an 11% error if readings are made at 650 nm.
Citation and/or Web Resources
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. J. Biol. Chem. 1951; 265:193