By Yan Zhang, Molecular Devices Corporation, 1311 Orleans Dr., Sunnyvale, CA 94089.
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.
NanoOrange Protein Quantitation Kit from Molecular Probes (catalog number (N-6666)
Protein for standard curve (note: BSA is supplied with the kit)
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)
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 the left panel of the screen and set up all the appropriate parameters for the assay: wavelength (EX 485, EM 590 and cutoff at 570), sensitivity of 16 (the higher the number, the higher the precision, but it will take longer to read the plate), assay plate type, wells to read, etc.
USING NANOORANGE PROTEIN KIT IN GEMINI XS, GEMINI EM AND SPECTRAMAX M2 MICROPLATE READERS
Note: Set the number of flashes to 30 when using the SpectraMax M2.
Step 4: Create a template of the assay showing where the plate blank and sample wells will be located on the microplate. (See Figure 3.)
Step 5: Create a template of the assay showing where standards, unknowns and plate blanks will be located on the microplate. To access the Template Editor, click the Template button in the Plate sections tool bar.
Prepare the assay
The method for this assay follows the instructions in the product information sheet for NanoOrange from Molecular Probes, except the assay volume is proportionately reduced from 2.0 ml to 180 l to fit the 96-well microplate format.
Step 1: Dilute the concentrated Component B (NanoOrange protein quantitation diluent) 10x in distilled water (one part Component B to nine parts distilled water).
Step 2: Dilute the concentrated Component A (NanoOrange protein quantitation reagent) 500x with the working concentration (1x) NanoOrange protein quantitation diluent to make the NanoOrange working solution.
Note: This should be done just prior to the experiment (the working solution should be used within a few hours of preparation). Only prepare enough of the NanoOrange working solution for the number of samples (including standards, unknowns, blanks and any other controls) in the assay.
Protect the NanoOrange working solution from light by placing it in an opaque bottle, by wrapping a non- opaque container in aluminum foil or by placing the solution in a dark cupboard or drawer. It is important to protect the working solution from light to prevent photodegradation of the reagent.
Step 3: Dilute 2 mg/ml BSA into 10 g/ml and 1 g/ml stock solution with the NanoOrange working solution.
Step 4: The reference curve and unknowns are prepared using the NanoOrange working solution. The standard curve shown in this application note was prepared using a 10 g/ml and 1 g/ml stock of BSA. The volumes given in this table are enough to prepare 5 replicates of each concentration. You may want to adjust the volumes, depending on the number of replicates you want to run. Mix the appropriate BSA with the NanoOrange working solution into eleven brown (for protection from light) eppendorf tubes as described in the following table:
Step 5: Incubate samples at 95˚C for 10 minutes (protected from light), then cool to room temperature (approximately 20 minutes), again keeping samples protected from light.
Step 6: Transfer 180 l of each sample to the appropriate well of the microplate. Triplicates of the BSA concentration were made for this assay plate.
Read the microplate
Step 1: Place the microplate in the reader.
Step 2: Click SoftMax Pros Read button. The microplate reader will read the plate and the relative fluorescence units will be displayed in the Plate section.
Analyze the data
Step 1: After the microplate has been read, the RFUs will be displayed in the Plate section. The data will be analyzed in the Group tables that you created while setting up the template (see the section Setting up the instrument and software earlier in this document).
Step 2: Plot the mean RFUs of the samples versus the BSA concentrations. (See Figure 4.)
Step 3: Choose the appropriate curve fit from the drop-down Curve Fit menu in the Graph sections tool bar. When plotting the standard curve, a linear curve fit was used.
The following standard curve was obtained from Molecular Devices Gemini XS system using the Molecular Probes, Inc. NanoOrange Protein Quantitation Kit with the BSA standard provided in the kit. (See Figure 5.) The low end of the standard curve is shown in Figure 6 with a linear curve fit. Identical data and curves from Gemini EM and SpectraMax M2 are not shown here.
The NanoOrange assay from Molecular Probes when run on a Gemini XS, Gemini EM or SpectraMax M2 microplate reader with SoftMax Pro software is a quick, sensitive detection method for proteins. The analysis capabilities of SoftMax Pro provide quantitation in an easy-to-read, usercustomizable report format.
Molecular Probes Data Sheet MP 6666 07/16/96: NanoOrange Protein Quantitation Kit (N-6666).