Hartree-Lowry and Modified Lowry Protein Assays

Considerations for use

The Lowry assay (1951) is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and so on. The bicinchoninic acid (BCA) assay is based on the same princple and can be done in one step, therefore it has been suggested (Stoscheck, 1990) that the 2-step Lowry method is outdated. However, the modified Lowry is done entirely at room temperature. The Hartree version of the Lowry assay, a more recent modification that uses fewer reagents, improves the sensitivity with some proteins, is less likely to be incompatible with some salt solutions, provides a more linear response, and is less likely to become saturated. The HartreeLowry assay will be described first.

Principle
Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue.

Equipment
In addition to standard liquid handling supplies a spectrophotometer with infrared lamp and filter is required. Glass or polystyrene (cheap) cuvettes may be used.

Procedure - Hartree-Lowry assay

Reagents
1. Reagent A consists of 2 gm sodium potassium tartrate x 4 H20, 100 gm sodium carbonate, 500 ml 1N NaOH, H20 to one liter (that is, 7mM Na-K tartrate, 0.81M sodium carbonate, 0.5N NaOH final concentration). Keeps 2 to 3 months. 2. Reagent B consists of 2 gm 2 gm sodium potassium tartrate x 4 H20, 1 gm copper sulfate (CuSO4 x 5H20), 90 ml H20, 10 ml 1N NaOH (final concentrations 70 mM Na-K tartrate, 40 mM copper sulfate). Keeps 2 to 3 months. 3. Reagent C consists of 1 vol Folin-Ciocalteau reagent diluted with 15 vols water.

Assay

1. Prepare a series of dilutions of 0.3 mg/ml bovine serum albumin in the same buffer containing the unknowns, to give concentrations of 30 to 150 micrograms/ml (0.03 to 0.15 mg/ml). 2. Add 1.0 ml each dilution of standard, protein-containing unknown, or buffer (for the reference) to 0.90 ml reagent A in separate test tubes and mix. 3. Incubate the tubes 10 min in a 50 degrees C bath, then cool to room temperature. 4. Add 0.1 ml reagent B to each tube, mix, incubate 10 min at room temperature. 5. Rapidly add 3 ml reagent C to each tube, mix, incubate 10 min in the 50 degree bath, and cool to room temperature. Final assay volume is 5 ml. 6. Measure absorbance at 650 nm in 1 cm cuvettes.

Analysis
Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

Procedure - modified Lowry (room temperature)

Reagents
1. Dissolve 20 gm sodium carbonate in 260 ml water, 0.4 gm cupric sulfate (5x hydrated) in 20 ml water, and 0.2 gm sodium potassium tartrate in 20 ml water. Mix all three solutions to prepare the copper reagent. 2. Prepare 100 ml of a 1% solution (1 gm/100 ml) of sodium dodecyl sulfate (SDS). 3. Prepare a 1 M solution of NaOH (4 gm/100 ml). 4. For the 2x Lowry concentrate mix 3 parts copper reagent with 1 part SDS and 1 part NaOH. Solution is stable for 2-3 weeks. Warm the solution to 37 degrees C if a white precipitate forms, and discard if there is a black precipitate. Better, keep the three stock solutions, and mix just before use. 5. Prepare 0.2 N Folin reagent by mixing 10 ml 2 N Folin reagent with 90 ml water. Kept in an amber bottle, the dilution is stable for several months.

Assay
1. Dilute samples to an estimated 0.025-0.25 mg/ml with buffer. If the concentration can't be estimated it is advisable to prepare a range of 2-3 dilutions spanning an order of magnitude. Prepare 400 microliters each dilution. Duplicate or triplicate samples are recommended. 2. Prepare a reference of 400 microliters buffer. Prepare standards from 0.25 mg/ml bovine serum albumin by adding 40-400 microliters to 13 x 100 mm tubes + buffer to bring volume to 400 microliters/tube. 3. Add 400 microliters of 2x Lowry concentrate, mix thoroughly, incubate at room temp. 10 min.

4. Add 200 microliters 0.2 N Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be accomplished quickly to avoid decomposition of the reagent before it reacts with protein. Incubate for 30 min. more at room temperature. 5. Use glass or polystyrene cuvettes to read the absorbances at 750 nm. If the absorbances are too high, they may be read at 500 nn.

Comments
Recording of absorbances need only be done within 10 min. of each other for this modified procedure, whereas the original Lowry required precise timing of readings due to color instability. This modification is less sensitive to interfering agents and is more sensitive to protein than the original. As with most assays, the Lowry can be scaled up for larger cuvette sizes, however more protein is consumed. Proteins with an abnormally high or low percentage of tyrosine, tryptophan, or cysteine residues will give high or low errors, respectively.

References

Lowry, OH, NJ Rosbrough, AL Farr, and RJ Randall. J. Biol. Chem. 193: 265. 1951. Oostra, GM, NS Mathewson, and GN Catravas. Anal. Biochem. 89: 31. 1978. Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). Hartree, EF. Anal Biochem 48: 422-427 (1972). Another Lowry

Reagents A. 2% Na2CO3 in 0.1 N NaOH B. 1% NaK Tartrate in H2O C. 0.5% CuSO4.5 H2O in H2O D. 48 mL of A, 1 mL of B, 1 mL C E. Phenol Reagent - 1 part Folin-Phenol [2 N] : 1 part water [Reagents A, B and C may be stored indefinitely] BSA Standard - 1 mg/ mL Bovine Serum Albumin: 5 mg in 5 mL of water [1 g / l]. Freeze 1 mL aliquots. Procedure [Run triplicate determination for all samples.]

1. Set up eleven sets of three 16 x 150 mm test tubes in rack. 2. Add BSA [0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 l] to these tubes. 3. Add 2 mL of solution D to each test tube. 4. Incubate for 10 minutes at room temperature. 5. Add 0.2 mL of dilute Folin-phenol solution to each tube. 6. Vortex each tube immediately. 9. Incubate at room temperature for 30 minutes. 10. Determine absorbance of each sample at 600 nm. 11. Plot absorbance vs mg protein to obtain standard curve. 12. Set up triplicate assays for all "unknowns".

Figure 6. Protein concentrations of a crude homogenate and a 45-65% pellet.

Lowry Method: A Protein Assay

Although Lowry method is not good enough compared to Bradford and Bicinchoninic Acid method for protein quantitation, but it is still widely used due to its acceptability to estimate protein in almost all circumstances in which protein mixtures or crude extracts are involved. Lowry method is based on the conversion of Cu2+ to Cu+ under alkaline conditions. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. Sensitivity of the method is down to about 0.01 mg of protein /mL, and is best used on solutions with concentrations in the range 0.01-1.0 mg/mL of protein. If your sample contains protein less than 0.01, then you can used Bradford method (it can be used for measuring between 1 and 10 micrograms of protein, for microassay) and Bicinchoninic Acid method (it can be used for measuring between 0.5 and 10 micrograms of protein/mL, for microassay).

Material 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) CuSO45H2O in distilled water. Solution C: 2% (w/v) sodium potassium tartrate in distilled water. 2. 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 20C. Prepare standards by diluting the stock solution with distilled water. You can make the solutions with several series concentration, for example 0; 10; 20; 50; 100; 200; 500; 1000; 2000 micrograms/mL. The Method are: 1. To 0.1 mL of sample or standard, add 0.1 mL of 2 N NaOH. Hydrolyze at 100C 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. o The reaction is very pH dependent, and it is therefore important to maintain the pH between 10 and 10.5. Therefore, take care when analyzing samples that are in strong buffer outside this range. o The incubation period is not critical and can vary from 10 min to several hours without affecting the final absorbance. 3. Add 0.1 mL of Folin reagent, using a vortex mixer, and let the mixture stand at room temperature for 3060 min (do not exceed 60 min). The vortex-mixing step is critical for obtaining reproducible results. The Folin reagent is reactive only for a short time under these alkaline conditions, being unstable in alkali, and great care should therefore be taken to ensure thorough mixing. 4. Read the absorbance at 750 nm if the protein concentration was below 500 micrograms/mL or at 550 nm if the protein concentration was between 100 and 2000 micrograms/mL. 5. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations. o The assay is not linear at higher concentrations. Ensure that you are analyzing your sample on the linear portion of the calibration curve. o A set of standards is needed with each group of assays, preferably in duplicate. Duplicate or triplicate unknowns are recommended. One disadvantage of the Lowry method is the fact that a range of substances interferes with this assay, including buffers, drugs, nucleic acids, and sugars. In many cases, the effects of these agents

can be minimized by diluting them out, assuming that the protein concentration is sufficiently high to still be detected after dilution. You can also increase Lowry methods sensitivity by doing these two following things: 1. If the Folin reagent is added in two portions, vortex-mixing between each addition, a 20% increase in sensitivity is achieved. 2. The addition of dithiothreitol 3 min after the addition of the Folin reagent increases the sensitivity by 50%.