| Contributor: |
The Laboratory of Andy Golden at the National Cancer Institute
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| This protocol describes the Lowry method for protein concentration determination. |
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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)
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| BSA Stock Solution |
| 1 mg/ml Bovine Serum Albumin Fraction V
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| Phenol Mix |
| 50% Folins Reagent diluted in ddH2O
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| Lowry Solution |
| 200 μl of Lowry A
10 ml of Lowry B
Make up this solution just before performing the assay.
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| Lowry B |
| 0.1 N NaOH
2% Na2CO3 anhydrous
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| Lowry A |
| 2% (w/v) Sodium Tartrate
1% (w/v) Copper Sulfate Pentahydrate
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Sodium Carbonate Anhydrous
Copper Sulfate Pentahydrate
Sodium Tartrate
Sodium Hydroxide
Bovine Serum Albumin
Folins Reagent
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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.
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Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. J. Biol. Chem. 1951; 265:193
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