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A mixture of Cu2‡ _{sulfate and sodium-potassium tartrate reacts with}

proteins. Then Folin-Ciocalteu reagent is added. A blue-purplish color forms that is measured at 750 nm (A750). The Lowry method is reviewed by

Lane (7) and Peterson (8,9). The following procedure is based on descriptions from Refs. 1 and 7. Preparation of the Folin-Ciocalteu reagent (4) is described in Method 2.

Method 1

Analysis of soluble proteins using the Lowry method. Reagents

1. Sodium carbonate (Na2CO3)

2. Sodium hydroxide (0.1 M) 3. Copper sulfate …CuSO4
5H2O†

4. Sodium potassium tartrate 5. Folin-Ciocalteu reagent 6. Bovine serum albumin Procedure

Prepare stock solutions for reagents A, B, and E as described below. Mix reagents A and B in a volume ratio of 50:1 to produce reagent C, which is a copper sulfate solution stabilized with tartrate. Reagent A (2% sodium carbonate in 0.1 N sodium hydroxide). Dissolve

sodium carbonate (2 g) in 100 mL of 0.1 M sodium hydroxide. Reagent B (0.5% copper sulfate in 1.0% sodium-potassium tartrate).

Add 0.5 g of copper sulfate and 1.0 g of tartrate to 100 mL of distilled water.*

Reagent C (alkaline copper tartrate). Mix 50 mL of reagent A and 1 mL of reagent B. Prepare daily.

Reagent E (Folin-Ciocalteu reagent). Dilute the commercial (2 N) reagent 1:1 with distilled water.

Standard protein assay. Mix 0.2 mL of protein sample (5±100 mg){ with 1 mL of reagent C. Incubate at room temperature for 10 minutes. Now add 0.1 mL of reagent E and mix immediately. After 30

* Copper sulfate and tartrate salts have a low solubility in alkali. Dissolve these separately in a small volume of water before adding to reagent A.

{ The prescribed reagent volumes lead to a 6.5-fold dilution of protein standards. The

concentration of protein should be 20±500 mg mL 1_{, leading to a ``within cuvette'' concentration}

minutes take A750 readings or else A500 readings for strongly

concentrated solutions.

The Folin-Ciocalteu reagent is now widely available. The commercial reagent is stable for months at room temperature. There seems little justi®cation for preparing reagent E in house. In the absence of a commercial supplier, the Folin-Ciocalteu regent may be prepared as follows. Method 2

Preparation of the Folin-Ciocalteu reagent Reagents

1. Sodium tungstate …Na2Wo4? 2H20† 100 g

2. Sodium molybdate …Na2MO4? 2H2O† 25 g

3. Water 700 mL

4. Phosphoric acid (85% w/w) 50 mL 5. Hydrochloric acid (conc.) 100 mL Procedure

Add the preceding compounds and some antibumping granules to a 1.5-L ¯ask. Fit a condenser and re¯ux gently for 10 hours in a fume cupboard. Switch off the gas or electric heater.

Remove the condenser and add (a) 150 g of lithium sulfate and (b) 50 mL of water followed by (c) a few drops of liquid bromine. Bring the mixture to a boil for 15 minutes to remove excess bromine. Allow to cool, make up the volume to 1L with distilled water, and ®lter. The ®nished reagent, which should have no green tint, should be stored in an amber bottle.

Reference 1 is one of the most often cited papers in analytical biochemistry.* It is one of the scienti®c papers for which the description classic is probably deserved. Much of what is known about the Lowry method was anticipated in the original publication 50 years ago. A number of investigators have modi®ed the Lowry assay to improve its performance characteristics. Bensadoun and Weinstein (10) reacted protein samples with sodium deoxycholate and trichloroacetic acid (TCA) before performing the Lowry assay. This pretreatment effectively frees protein samples from TCA- soluble compounds, thereby improving accuracy. When 5 and 50 mg of protein were precipitated with TCA, the recovery ranged from 7 to 91%. Pretreatment with DOC led to the quantitative recovery of protein no matter its initial concentration. With samples containing high concentra- tions of the interferences, two cycles of precipitation were used. The initial

protein-DOC precipitate was resuspended in distilled water and reprecipi- tated for a second time. Peterson (2) used DOC-TCA precipitation to develop an assay, now available in kit form (Sigma-Aldrich Ltd.), that rendered the classical Lowry method obsolete.*

Method 3

Peterson's modi®cation of the Lowry protein assay (2,8,9) Reagents

1. Copper sulfate

2. Sodium potassium tartrate 3. Sodium carbonate

4. Sodium dodecyl sulfate (SDS) stock solution (5% w/v) 5. Sodium deoxycholate solution (0.15% w/v)

6. Sodium hydroxide stock solution (0.8 M) 7. Trichloroacetic acid (72% w/v)

8. Folin-Ciocalteu reagent (2 N)

9. Bovine serum albumin (BSA) (0.5 mg mL 1_{) as protein standard.}

Add 1 mg mL 1_{sodium azide as preservative and store frozen as}

small aliquots. Procedurey

Copper-tartrate-carbonate (CTC) stock solution. Dissolve copper sulfate (0.1 g) as well as sodium potassium tartrate (0.2 g) each in about 10 mL of distilled water. Add both solutions to sodium carbonate (10 g in 50 mL of distilled water) and make up to a ®nal volume of 100 mL.

Preparation of reagent A. Mix CTC, SDS, and sodium hydroxide stock solutions in a volume ration of 1:2:1. Reagent A is stable at room temperature for 2±3 weeks. Refrigeration at 48C will double the useful life. SDS precipitates in the refrigerator. Redissolve by holding the reagent bottle under a running hot-water tap.

Preparation of reagent B. Dilute commercial 2 N Folin-Ciocalteu reagent 1:1 with distilled water. This is stable for many months at room temperature.

Protein analysis. Add suf®cient water to bring the protein sample (5± 100 mg) volume to 1 mL. Now, add 0.1 mL of DOC solution, followed 10 minutes later with 0.1 mL of TCA solution. Centrifuge the mixture at 10,000 g (mark 12 on a microcentrifuge) for 10 minutes. Decant the supernatant and place the upturned Eppendorf

* Peterson's modi®cation of the Lowry assay had received 6130 citations as of June 2001. { It is convenient to employ (1.5-mL) microcentrifuge (Eppendorf) tubes for this procedure.

tubes over tissue paper to drain. The precipitate of DOC-protein may just be seen as a gray plaque on the wall of the microcentrifuge tube.

Add reagent A (1 mL) and mix gently to redissolve the protein precipitate. After 10 minutes add reagent B and allow to stand for 30 minutes. Take A750 readings using a 1-cm (1.7 mL capacity)

disposable plastic cuvette.

The distinctive features of Peterson's method can be summarized as follows: 1. The increased concentration of copper tartrate increases the

stability of the Lowry reagents from 24 hours to 2 weeks. 2. SDS (1% w/v) reduces interferences from nonionic detergents and

dissolves membrane proteins.

3. Protein precipitation with DOC-TCA eliminates nonprotein interferences.

4. The precipitation step concentrates samples such that &1m g of protein can be detected accurately.

3. CHEMISTRY OF THE LOWRY ASSAY