Section 8- Protein A280
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
.
Pedestal Reconditioning
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 measurement
surface aggressively with a dry laboratory wipe 30-40 times. This will “re-condition” the surface allowing the liquid sample
column to form. Alternatively, use the NanoDrop Pedestal Reconditioning Compound (PR-1) as a rapid means of
reconditioning the pedestals when the surface properties have been compromised and liquid columns break during
measurement. Additional information about the PR-1 kit may be found at
www.nanodrop.com.
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
Detection
Limit
Approx. Upper
Limit
Typical Reproducibility
(minimum 5 replicates)
(SD= mg/ml; CV= %)
Purified BSA
0.10 mg/ml
100 mg/ml
sample range 0.10-10 mg/ml:
±
0.10 mg/ml
sample range >10mg/ml:
±
2%
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.
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