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A
TD-700 Laboratory Fluorometer Method for
NanoOrange™
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 µg/mL), the Bradford assay (1 µg/mL), the
Lowry assay (1 µg/mL), or 280 nm absorbance (50 µg/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 µg/mL to be accurately measured relative to a standard
curve (Figures 1 and 2).
Figure 1. Full-range calibration plot for bovine serum albumin (BSA) using
the TD-700 Fluorometer and the NanoOrange Protein Quantitation Kit.
Figure 2. Low-range calibration plot for bovine serum albumin (BSA) using
the TD-700 Fluorometer and the NanoOrange Protein Quantitation Kit.
Materials Required
- TD-700 Fluorometer with standard PMT
and 10 x 10 mm square cuvette adaptor (P/N 7000-009)
- Blue Mercury Vapor Lamp (P/N 10-089)
- Fluorescein filter kit (P/N 10-086R)
which includes 486 nm excitation filter (P/N 034-0486) and 510-700 nm
emission filter (P/N 10-109R-C).
- 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.
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 µL 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
µg/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
µg BSA per mL. If desired, serial dilutions can be made to create additional
standards ranging from 0.01 to 0.6 µg/mL, to fill out the standard curve
in the low range.
3.2.1 Prepare a 10 µg/mL solution
of BSA by diluting 30 µL 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 µg/mL BSA solution
to make 0, 1, 3, 6 and 10 µg/mL standards, as described in Table 1. If
desired, prepare 0.1, 0.3 and 0.6 µg/mL standards, as described in Table
1, by diluting a 1 µg/mL BSA solution [note B]. Prepare the 1 µg/mL BSA
solution by diluting 300 µL of 10 µg/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 µg/mL standards, as described in Table 1, by diluting a
0.1 µg/mL BSA solution [note B]. Prepare the 0.1 µg/mL BSA solution by
diluting 300 µL of 1 µg/mL BSA (made in section 3.2.2) into 2.70 mL of
1X NanoOrange working solution.
 Table
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
µg/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 µg/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 µg/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.
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.
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.
References
- Anal Biochem 150, 76 (1985)
- Anal Biochem 72, 248 (1976)
- J Biol Chem 193, 265 (1951)
- Scopes, R.K., Protein Purification,
Principles and Practice, 2nd Edition, Springer-Verlag (1987)
About Molecular Probes
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Email: order@probes.com
Mail: Molecular Probes, Inc., PO Box 22010, Eugene, OR 97402-0469, USA
Information on the scientific and technical
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Email: tech@probes.com
Internet: http://www.probes.com
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