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HOME > Protocols > Cell Biology > Protocol for Quantiation of Proteins using NanoOrange

Protocol for Quantiation of Proteins using NanoOrange

1. Introduction

Fluorometric quantitation of proteins in solution using the Turner BioSystems TD-700 Laboratory Fluorometer and Molecular Probes' NanoOrange™ Protein Quantitation Kit offers an unprecedented combination of convenience and sensitivity. Protein concentrations as low as 10 ng/mL can be measured. This level of sensitivity is much superior to spectrophotometric techniques such as the BCA method (0.5 g/mL), the Bradford assay (1 g/mL), the Lowry assay (1 g/mL), or 280 nm absorbance (50 g/mL). (1-4)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 95 C for ten minutes. Fluorescence can be measured as soon as the mixture has cooled to room temperature. Alternatively, samples can be read up to six hours after preparation with no loss in sensitivity, as long as samples are protected from light. The NanoOrange™ reagent is virtually nonfluorescent in aqueous solution, becoming strongly fluorescent at about 570—590 nm upon interaction with proteins, when excited at about 470—490 nm. Detection of the fluorescence using the TD-700 fluorometer equipped with a fluorescein filter kit allows protein concentrations from 10 ng/mL to 10 g/mL to be accurately measured relative to a standard curve (Figures 1 and 2).

Figure 1
Figure 1. Full-range calibration plot for bovine serum albumin (BSA) using the TD-700 Fluorometer and the NanoOrange™ Protein Quantitation Kit.

Figure 2
Figure 2. Low-range calibration plot for bovine serum albumin (BSA) using the TD-700 Fluorometer and the NanoOrange™ Protein Quantitation Kit.

2. Materials Required

  • TD-700 Fluorometer with standard PMT and 10 x 10 mm square cuvette adaptor (P/N 7000-009)
  • Fluorescein filter kit (P/N 10-086R) which includes 486 nm excitation filter (P/N 034-0486), 510-700 nm emission filter (P/N 10-109R-C), and two Blue Mercury Vapor Lamps (P/N 10-089).
  • 10 x 10 mm square polystyrene disposable cuvettes (P/N 7000-957)
  • NanoOrange™ Protein Quantitation Kit, supplied by Molecular Probes, Inc., Eugene, Oregon, catalog number N-6666. The kit contains 1.0 mL NanoOrange™ protein quantitation reagent (500X concentrate), 50 mL NanoOrange™ protein quantitation diluent (10X concentrate), and 0.5 mL bovine serum albumin (BSA) standard (2 mg/mL). The kit contents are sufficient for 200 assays using a 2 mL volume in a standard cuvette. Handling, storage, and the use of the reagents should be performed in accordance with the product information sheet supplied by Molecular Probes, Inc.

3. Experimental Protocol

3.1 Reagent Preparation

Dilute the concentrated NanoOrange™ protein quantitation diluent 10-fold in distilled water. For each assay, 2.5 mL of 1X protein quantitation diluent will be required. Just prior to running the experiment, dilute the NanoOrange™ protein quantitation reagent 500-fold into the 1X protein quantitation diluent.

For example, to prepare 50 mL of 1X NanoOrange™ working solution (enough for 20 assays), first prepare the 1X diluent by mixing 5 mL of the 10X diluent stock with 45 mL of distilled water; next add 100 L of the NanoOrange™ reagent and mix thoroughly. Protect the 1X NanoOrange™ working solution from photodegradation by storing it in an opaque bottle, covering it with foil or placing it in the dark. For best results, the working solution should be used within a few hours of its preparation.

3.2 Protein Standard Curve

A standard curve should be generated for converting sample fluorescence into protein concentration. Ideally, the protein type used for the standard curve should be the same as that which is used in the experiment; however, as with other protein assays, bovine serum albumin (BSA) serves as a convenient reference standard. The NanoOrange™ Kit includes a 2 mg/mL sample of BSA that can be used to prepare a standard curve. To serve as an effective control, the protein solution used to prepare the standard curve should also contain levels of contaminants similar to those present in the experimental samples [note A]. The reference standard curve is used not only to convert fluorescence to protein concentration, but also to control for any day-to-day readout variation of the fluorometer. The standard curve may be generated to cover the full assay range, 0-10 g/mL, or to cover a selected range. This section describes how to generate a simple standard curve with points corresponding to 0, 1, 3, 6 and 10 g BSA per mL. If desired, serial dilutions can be made to create additional standards ranging from 0.01 to 0.6 g/mL, to fill out the standard curve in the low range.

3.2.1 Prepare a 10 g/mL solution of BSA by diluting 30 L of the BSA standard into 5.97 mL of the 1X NanoOrange™ working solution prepared in section 3.1.

3.2.2 Dilute the 10 g/mL BSA solution to make 0, 1, 3, 6 and 10 g/mL standards, as described in Table 1. If desired, prepare 0.1, 0.3 and 0.6 g/mL standards, as described in Table 1, by diluting a 1 g/mL BSA solution [note B]. Prepare the 1 g/mL BSA solution by diluting 300 L of 10 g/mL BSA (made in section 3.2.1) into 2.70 mL of 1X NanoOrange™ working solution.

3.2.3 If desired, prepare 0.01, 0.03 and 0.06 g/mL standards, as described in Table 1, by diluting a 0.1 g/mL BSA solution [note B]. Prepare the 0.1 g/mL BSA solution by diluting 300 L of 1 g/mL BSA (made in section 3.2.2) into 2.70 mL of 1X NanoOrange™ working solution.

Table 1Table 1. Protocol for preparing a standard curve using BSA.

3.2.4 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.

3.2.5 After cooling, transfer 2.0 mL [note B] of the sample to a standard acrylic fluorescence cuvette and measure the fluorescence using the TD-700 Fluorometer installed with the blue mercity vapor lamp (P/N 10-089), excitation filter 034-0486, and emission filter 10-109R-C. Insert the most fluorescent sample first (10 g/mL protein) and calibrate the instrument sensitivity as directed in the TD-700 manual (press #2, calibrate). This procedure automatically optimizes the instrument sensitivity to match the fluorescence of the sample.

3.2.6 Measure the fluorescence of the remaining samples. To equalize any photobleaching effects, insert samples into the fluorometer for approximately equal time periods. The fluorescence value of the reagent blank (0 g/mL protein) may be subtracted from that of each sample. Corrected or uncorrected data may be used to generate a standard curve of fluorescence versus protein concentration (for example, see Figures 1 and 2).

 

 

3.3 Sample Analysis

3.3.1 Dilute the experimental protein solution in 1X NanoOrange™ working solution (prepared in section 3.1) to achieve a final volume of 2.5 mL [note B]. You may wish to use two or three different dilution factors for a given sample. Higher dilution factors will diminish levels of contaminants [note A]; however, extremely small sample volumes should be avoided as they are difficult to pipet 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 [note B] of the sample to a standard acrylic fluorescence cuvette and measure the fluorescence using the same instrument parameters as used in generating the standard curve (section 3.2.6). To equalize any photobleaching effects, insert samples into the fluorometer for similar time periods to those used for the standard curve measurements.

3.3.4 If the standard curve was plotted using blank-subtracted data (section 3.2.6), the reagent blank (0 g/mL protein) fluorescence value must also be subtracted from that of each of the samples. Determine the protein concentration of the sample from the standard curve generated in section 3.2.6.

4. Footnotes

[A] Various compounds known to contaminate protein preparations, including salts, detergents and reducing agents may interfere with the NanoOrange™ protein quantitation assay. Protein standard and blank samples should be prepared in solutions that match the composition of the unknown samples as closely as possible. The maximum tolerable concentrations for avoiding appreciable interference are approximately 10 mM for salts (including ammonium sulfate), 100 mM for reducing agents (DTT and b-mercaptoethanol) and 0.01% (w/v) for SDS. For other detergents (Tween-20 and Triton X-100), the tolerance level is lower (0.001% (w/v)). See Molecular Probes' product information sheet MP6666 for further details.

[B] Pipetting and sample handling are the largest sources of experimental error in the assay. Accurate volume measurements are essential when making up and transferring samples.

5. Warnings and Precautions

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.


6. References

  1. Anal Biochem 150, 76 (1985)
  2. Anal Biochem 72, 248 (1976)
  3. J Biol Chem 193, 265 (1951)
  4. Scopes, R.K., Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag (1987)

 

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