|List of methods
Direct absorbance measurement
absorbance at 280 nm
absorbance at 205 nm
set up an assay
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(Stoscheck, 1990) that the 2-step Lowry method is outdated.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.
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.
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
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.
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.
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)
- 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
- Prepare 100 ml of a 1% solution (1 gm/100 ml) of sodium dodecyl
- 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.
- 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.
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.
- 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).