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List of methods

Direct absorbance measurement

absorbance at 280 nm
absorbance at 205 nm
extinction coefficient

Colorimetric assays

set up an assay
spectrophotometry
modified Lowry
biuret
Bradford
Bicinchoninic Acid (Smith)

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princple and can be done in one step, therefore it has been suggested (Stoscheck, 1990) that the 2-step Lowry method is outdated.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 Hartree-Lowry 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

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.
Reagent B consists of 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.
Reagent C consists of 1 vol Folin-Ciocalteau reagent diluted with 15 vols water.

Assay

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).
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.
Incubate the tubes 10 min in a 50 degrees C bath, then cool to room temperature.
Add 0.1 ml reagent B to each tube, mix, incubate 10 min at room temperature.
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.
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

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.
Prepare 100 ml of a 1% solution (1 gm/100 ml) of sodium dodecyl sulfate (SDS).
Prepare a 1 M solution of NaOH (4 gm/100 ml).
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.
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

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.
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.
Add 400 microliters of 2x Lowry concentrate, mix thoroughly, incubate at room temp. 10 min.
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.
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).

Copyright and Intended Use
Visitors: to ensure that your message is not mistaken for SPAM, please include the acronym "Bios211" in the subject line of e-mail communications
Created by David R. Caprette (caprette@rice.edu), Rice University 24 May 95
Updated 12 Jun 15



Home	Laboratory Studies	Recordkeeping, Writing, & Data Analysis	Laboratory Methods
	Overview Microscope studies Flagella experiment Laboratory math Blood fractionation Gel electrophoresis Protein gel analysis Mitochondria Concepts/ theory	Overview Keeping a lab notebook Writing research papers Dimensions & units Using figures (graphs) Examples of graphs Experimental error Representing error Applying statistics	Overview Principles of microscopy Solutions & dilutions Protein assays Spectrophotometry Fractionation & centrifugation Radioisotopes and detection

List of methods Direct absorbance measurement absorbance at 280 nm absorbance at 205 nm extinction coefficient Colorimetric assays set up an assay spectrophotometry modified Lowry biuret Bradford Bicinchoninic Acid (Smith)	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 Hartree-Lowry 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 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. Reagent B consists of 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. Reagent C consists of 1 vol Folin-Ciocalteau reagent diluted with 15 vols water. Assay 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). 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. Incubate the tubes 10 min in a 50 degrees C bath, then cool to room temperature. Add 0.1 ml reagent B to each tube, mix, incubate 10 min at room temperature. 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. 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 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. Prepare 100 ml of a 1% solution (1 gm/100 ml) of sodium dodecyl sulfate (SDS). Prepare a 1 M solution of NaOH (4 gm/100 ml). 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. 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 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. 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. Add 400 microliters of 2x Lowry concentrate, mix thoroughly, incubate at room temp. 10 min. 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. 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).
	Copyright and Intended Use Visitors: to ensure that your message is not mistaken for SPAM, please include the acronym "Bios211" in the subject line of e-mail communications Created by David R. Caprette (caprette@rice.edu), Rice University 24 May 95 Updated 12 Jun 15