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8. Protein A280
Proteins, unlike nucleic acids, can exhibit considerable diversity. The A280 method is applicable to purified proteins exhibiting
absorbance at 280nm. It does not require generation of a standard curve and is ready for quantitation of protein samples at startup. This
module displays the UV spectrum, measures the protein's absorbance at 280 nm (A280) and calculates the concentration (mg/ml). Like
the Nucleic Acid module, it automatically switches to the 0.2 mm pathlength at very high concentrations of protein. Also analogous to
the Nucleic Acid module, the Protein A280 module displays and records 10 mm (1 cm) equivalent data on the screen and in the
archived data file.
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. That
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 for the liquid sample column to form.
Measurement Concentration Range
®
The NanoDrop
ND-1000 Spectrophotometer will accurately measure protein samples up to 100 mg/ml (BSA) without dilution. To do
this, the instrument automatically detects the high concentration and utilizes the 0.2mm pathlength to calculate the absorbance. A table
of concentration range and typical reproducibility is listed below.
Sample Type
Purified BSA
Unique Screen Features
Sample Type: There are six sample types (options) available for purified protein analysis and concentration measurement. All of the
options can be viewed by clicking the mouse while it is positioned within the 'Sample Type' box. The sample type (color-keyed) can be
selected by clicking on the preferred option or by scrolling through the selections using the up or down arrow keys located to the left of
the sample type box. A description of each sample type is given below.
Detection
Approx. Upper
Limit
Limit
0.10 mg/ml
100 mg/ml
A general reference setting based on a 0.1% (1 mg/ml) protein solution producing an Absorbance at 280 nm of
1.0 A (where the pathlength is 10 mm or 1 cm).
Bovine Serum Albumin reference. Unknown (sample) protein concentrations are calculated using the mass
extinction coefficient of 6.7 at 280 nm for a 1% (10 mg/ml) BSA solution.
Typical Reproducibility
(minimum 5 replicates)
sample range 0.05-10 mg/ml: ± 0.10 mg/ml
sample range >10mg/ml: ± 2%
8-1
Section 8- Protein A280
(SD= mg/ml; CV= %)
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