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Section 9- Proteins & Labels
9. Proteins & Labels
This software module can be used to determine protein concentration (A280nm) as well as fluorescent dye concentration (protein array
conjugates), or to measure the purity of metalloproteins (such as hemoglobin) using wavelength ratios.
Fluorescent Dye Selection
There are currently nine fluorescent dyes that are hard-coded for use with the Proteins and Labels module (see table below). Users can
also enter & save fluorescent dyes not coded within the ND-1000 software using the 'Dye/Chromophore Editor' button found in the main
menu.
Dyes can be selected using the scroll arrows or by highlighting the Dye 1 or Dye 2 box. The respective absorbance
wavelength,
extinction coefficient, and 280nm % corrections will be automatically utilized for measurement and concentration calculation. The
default settings from NanoDrop remain Dye 1 set to Cy3 and Dye 2 set to Cy5. In addition to the fluorescent dyes available from the
drop-down menu, an option entitled 'None' is also available. Selecting 'None' disables the respective calculations & numeric displays
corresponding to that dye.
Note: Please refer to the dye manufacturer for the appropriate correction factors for user entered dyes.
Sample Volume Requirements
Some proteins are hydrophobic and others hydrophilic giving rise to variable surface tension in the samples 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 for the liquid sample column to form.
Measurement Concentration Range
®
The NanoDrop
ND-1000 Spectrophotometer will accurately measure protein samples up to 20 mg/ml (BSA) without dilution. A table of
concentration range and typical reproducibility is listed below.
Approx.
Typical Reproducibility
Sample
Detection
Upper
(minimum 5 replicates)
Type
Limit
Limit
(SD= mg/ml; CV= %)
sample range 0.05-10 mg/ml: ± 0.10 mg/ml
Purified BSA
0.10 mg/ml
20 mg/ml
sample range >10mg/ml: ± 2%
sample range 0.20-4.0 pmol/ul: ± 0.20 pmol/ul
Cy3
sample range >4.0 pmol/ul: ± 2%
0.2uM
100uM
Unique Screen Features
9-1
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