GENERAL METHODS >
45
Absorbance
PathCheck
®
applied to measurement of
protein solutions in the SpectraMax
®
Plus microplate spectrophotometer
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>
Molecular Devices MAXline Application Note #26.
Protein concentration is commonly
estimated by measuring the absorbance
at 280 nm (A
280
) and calculating the
concentration using the extinction
coefficient of the protein. The proce-
dure is feasible because extinction coef-
ficients are typically obtained using
standard cuvettes with a fixed 1 cm
pathlength. Extinction-based protein
assays can also be done in microplates,
but they have not been widely used
because of the inconvenience of com-
pensating for variable pathlengths of
samples in microplate wells. The intro-
duction of the Molecular Devices'
SpectraMax Plus microplate spectro-
photometer marks the first time that
photometric measurements made in a
microplate format can be automatically
normalized such that the resultant val-
ues are identical to those obtained
using the corresponding solutions in a
standard 1 cm pathlength cuvette. This
application note outlines the use of the
pathlength correction feature "Path-
Check", compares absorbance results
obtained in cuvettes and microplate,
and gives an example in which Path-
Check is applied to column chromato-
graphic fractions and the elution
profile is displayed.
Fluorescence
Using the NanoOrange Protein Quan-
titation Kit in the Max microplate
fluorometer
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>
Molecular Devices MAXline Application Note #22.
Summary. This application note
describes how to use the NanoOrange
®
Protein Quantitation Kit from Molecu-
lar Probes in the Max fluorescence
microplate reader with SoftMax
®
Pro
software from Molecular Devices Cor-
poration. This assay is much more sensi-
tive than traditional photometric
methods such as A
280
, BCA, Bradford,
or Lowry assays in the microplate for-
mat. The dynamic range of this assay in
microplate format is 100 ng/mL to
10 µg/mL. The assay is read with an
excitation wavelength of 485 nm and an
emission wavelength of 590 nm. Both
the excitation and emission filters are
standard on the Max fluorescence
microplate reader. The kit from Molecu-
lar Probes contains sufficient reagent for
2000 assays using a 200 µL volume. The
data presented in this application note
confirm the dynamic range and lower
limit of detection described by Molecu-
lar Probes in their Application Note
(MP 6666). The data presented here are
preliminary and do not represent a fully
optimized assay.
protein quantitation
Summary :
Extinction-based protein assays can also be done in microplates, but they have not been widely used because of the inconvenience of com- pensating for variable pathlengths of samples in microplate wells. This application note describes how to use the NanoOrange ® Protein Quantitation Kit from Molecu- lar Probes in the Max fluorescence microplate reader with SoftMax ® Pro software from Molecular Devices Cor- poration.
Tags :
protein,application,note,using,deices,molecular,assay,280,lar,standard,assays,max,pathlength