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11. Protein Lowry
The Modified Lowry Protein Assay is an alternative method for determining protein concentration based on the widely used and cited
Lowry procedure for protein quantitation. Like the BCA and Bradford Assays, the Modified Lowry Assay requires standard curve
generation each time it is run, before unknown proteins can be measured. The Modified Lowry procedure involves reaction of protein
with cupric sulfate in alkaline solution, resulting in formation of tetradentate copper-protein complexes. The Folin-Ciocalteu Reagent is
effectively reduced in proportion to the chelated copper-complexes resulting in a water-soluble blue product that is measured at 650 nm
and normalized at 405 nm. Pre-formulated reagents, utilized in the assay, are available in kit form from numerous manufacturers.
Follow their recommendations when mixing the respective reagents at the time the assay is to be performed.
Sample Volume Requirements
Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the sample to be measured. Additionally
the presence of surfactants or detergents in reagents, such as the Bradford reagent, can significantly alter surface tension. This
occurrence can be overcome without affecting the sample's absorbance by using a larger sample volume. A 2 ul sample size is
recommended for protein measurements.
Special Cleaning Requirements for Proteins
Proteins and solutions containing surfactants are known to "un-condition" the measurement pedestal surfaces so that the liquid column
does not form. If this occurs, "buff" the measurement pedestal surfaces by rubbing each with a dry laboratory wipe 15-20 times. This
will "re-condition" the surface allowing the liquid sample column to form.
Measurement Concentration Range
®
On the NanoDrop
ND-1000 Spectrophotometer, the Modified Lowry assay can run from ~0.20 mg/ml up to 8.0 mg/ml.
Assay
Type
Modified Lowry
Modified Lowry Kits, Protocols, and Sample Preparation
Commercial Modified Lowry Protein kit manufacturers typically outline procedures for their assays.
Modified Lowry assay – using a 5:1 reagent / sample volume ratio.
•
To accurately prepare Standards, we suggest using a minimum sample volume of 20 ul and 100ul of Modified Lowry reagent.
Using the same pipettor for both volumes will eliminate any pipette-to-pipette accuracy differences.
•
After the initial 10 minute room temperature (RT) incubation period, add 10 ul of the Folin –Ciocalteu reagent and incubate for
30 minutes at RT.
In addition to the kit reagents, protein standards (BSA) for generating a standard curve are also provided by the manufacturer for the
Modified Lowry method. Follow the manufacturer's protocol for the assay including recommended incubation times and temperature.
Additionally, use the respective standard (e.g., BSA) and dilutions that cover the analytical range (mg/ml) of interest. Note: Since the
ND-1000 can measure higher protein concentrations, you may need to supply your own protein standards at higher concentrations than
provided by the manufacturer.
Unique Screen Features
View Standard Curve (F8): selecting this button allows the user to view the Standard Curve at any time.
Sample Type: choices are Reference, Standards 1-5, and Sample. The software will guide you to measure your reference, then at
least one standard before allowing measurement of samples.
Replicate #: counter for tracking replicate number during reference and standard measurement.
Reset Window (F11): clears all replicates of all standards.
Reset This Std (F12): clears all replicates of the selected standard.
Absorbance at 650nm: the Cu-complex's absorbance at 650 nm for the 1 mm pathlength.
Cursor λ and Absorbance: this feature allows the user to adjust the cursor wavelength and view the corresponding absorbance. The
cursor wavelength can be set by the user. Note: the user-selected wavelength and absorbance are not utilized in any calculations.
mg/ml: the concentration of the sample (unknown).
Approx.
Approx.
Lower
Upper
Limit
Limit
0.2 mg/ml
4.0 mg/ml
11-1
Section 11-Protein Lowry
Typical Reproducibility
(minimum 5 replicates)
(CV= %)
± 2% (over entire range)
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