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TBS-380 Mini-Fluorometer Method for Protein Quantitation Using|
Fluorometric quantitation of proteins in solution using the Turner BioSystems TBS-380 Mini-Fluorometer and Molecular Probes' NanoOrange™ Protein Quantitation Kit offers a combination of convenience and sensitivity. Protein concentrations as low as 100 ng/mL can be measured. This level of sensitivity is superior to spectrophotometric techniques such as the Bradford assay (1 µg/mL), the Lowry assay (1 µg/mL), or 280-nm absorbance (50 µg/mL).1-3 The NanoOrange™ assay also shows less protein-to-protein variability than the Bradford assay.
To perform a
protein assay, the protein sample is simply added to the NanoOrange™ reagent
in a specialized diluent and this mixture is heated at
2. MATERIALS REQUIRED
Mini-Fluorometer (P/N 3800-003).
Figure 1 and 2. Full-range calibration plot and close up of low range plot for bovine serum albumin (BSA) using the TBS-380 Mini-Fluorometer and the NanoOrange™ Protein Quantitation Kit.
3. EXPERIMENTAL PROTOCOL
3.2.1 Prepare a 20 µg/mL solution of BSA by diluting 60 µL of the BSA standard into 5.94 mL of the 1X protein quantitation diluent prepared in Section 3.1.
Dilute the 20µg/mL BSA solution to make a series of BSA standard solution at 2X
final concentration as described in Table 1. Mix equal volume of the 2X
NanoOrange™ working solution with each 2X BSA standard solution.
3.2.3 Incubate samples at 90°C to 96°C for 10 minutes, protected from light. After heating, cool to room temperature for 20 minutes, protected from light.
3.2.4 After cooling, transfer at least 2.0 mL[B] of the sample to a polystyrene cuvette or 50 mL of the sample to a Minicell cuvette, and measure the fluorescence using the TBS-380 Mini-Fluorometer. Set-up the fluorometer as per instructions in the User's manual. Power up the instrument by pressing the [ON/OFF] button. Use the [A/B] button to toggle to the "Blue" channel. Press [STD VAL] to program in the concentration of your calibration standard. Use the up and down arrows to change the concentration value. When ready, press the [CAL] button to start the calibration. The TBS-380 Mini-Fluorometer's screens will lead you through the calibration process.
3.2.5 Measure the fluorescence of the remaining standards. Use the data from the standards to generate a standard curve of fluorescence versus protein concentration. (Figures 1 and 2).
3.3 Sample Analysis
3.3.1 Dilute unknow protein samples to a desired volume in 1X protein quantitation diluent (prepared in section 3.1). Mix equal volume of the sample with the 2X NanoOrange working solution. You may wish to use two or three different dilution factors for a given sample. Higher dilution factors will diminish levels of contaminants[A]; however, extremely small sample volumes should be avoided, as they are difficult to pipette accurately.
3.3.2 Incubate samples at 90°C to 96°C for 10 minutes, protected from light. After heating, cool to room temperature for 20 minutes, protected from light.
3.3.3 After cooling, transfer 2.0 mL[B] of the sample to a 10x10 mm cuvette or 50 mL to a Minicell cuvette and measure the fluorescence using the same
instrument parameters as used in generating the standard curve (Section 3.2.4).
3.3.4 Determine the protein concentration of the sample from the standard curve generated in Section 3.2.5.
4. PATENTS AND TRADEMARKS
The NanoOrange™ Protein Quantitation Reagent is the subject of patent applications filed by Molecular Probes, Inc. and is not available for commercial uses without a specific agreement from Molecular Probes, Inc. NanoOrange is a trademark of Molecular Probes, Inc. Triton is a registered trademark of Rohm & Haas, Inc. Tween is a registered trademark of ICI Americas, Inc.
1. Anal Biochem
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