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Using the NanoOrange protein kit in the Gemini XS, Gemini EM and SpectraMax M2 microplate readers (MaxLine Application Note #22)

By Yan Zhang, Molecular Devices Corporation, 1311 Orleans Dr., Sunnyvale, CA 94089.


INTRODUCTION
This application note describes how to use the NanoOrange Protein Quantitation Kit from Molecular Probes in the Gemini XS, Gemini EM and SpectraMax M2 microplate readers with SoftMax Pro software from Molecular Devices. This assay is much more sensitive than traditional photometer methods, such as absorbance 280, BCA, Bradford or Lowry assays, in the microplate format. The dynamic range of this assay in microplate format is 10 g/ml to 10 ng/ml. The data presented in this application note confirms the dynamic range and lower detection limit described by Molecular Probes in their application note (MP 6666).

The assay is read with an excitation wavelength of 485 nm, an emission wavelength of 590 nm and cutoff at 570 on the Gemini XS spectrofluorometer. The dual monochromators in Gemini XS allow the selection of any wavelength in 1 nm increments.


MATERIALS
NanoOrange Protein Quantitation Kit from Molecular Probes (catalog number (N-6666)
Protein for standard curve (note: BSA is supplied with the kit)
Distilled water
Heating block at 90-96˚C
Plastic tubes (we used 1 ml eppendorf tubes)
Black microplates (wall and bottom of plate)
Gemini XS fluorescence microplate reader
SoftMax Pro software (version 4.5 or later)


METHODS

Set up the instrument and software
Step 1: Turn on your microplate reader

Step 2: Launch SoftMax Pro software. Initial setup screen will show. (See Figure 1.)

Step 3: Click Set Up and a screen will appear. (See Figure 2.) First, choose Fluorescence as the read type, then go to each option in
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