Living Colors rAcGFP1

Living Colors rAcGFP1
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A Modulus™ Microplate Fluorometer Method for
Quantitation of Living Colors®
rAcGFP1 in Gene Expression and
Protein Visualization Studies
From Clontech:
INTRODUCTION
®
Clontech Living Colors recombinant green
fluorescent protein from Aequorea
coerulescens (rAcGFP1) is a new alternative
to monomeric enhanced GFP (EGFP). The
spectral properties of rAcGFP1 closely
resemble those of EGFP; rAcGFP1,
excitation maximum is 475 nm and emission
maximum is 505 nm as compared to
484/510 nm for EGFP. In addition, the
rAcGFP1 protein has 94% homology to
EGFP at the amino acid level and is very
stable, allowing flurorescence examination
over an extended period of time. rAcGFP1
can be used as a fluorescent marker for
gene expression reporter assays in
organisms varying from bacteria to higher
plants and animals.
This application note describes a method
successfully used to quantitate rAcGFP1
using the Turner BioSystems Modulus™
Microplate Fluorometer. This instrument
can detect concentrations of rAcGFP1
ranging from as low as 10 pg/µL and up to
10 ng/µL in 96-well microplate format.
MATERIALS REQUIRED
From Turner BioSystems:
•
•
•
Modulus™ Microplate Fluorometer
(P/N 9300-000) or Modulus™ Microplate
Multimode Reader (9300-002)
Fluorescence Optical Kit – Blue,
460/515 - 580 nm (P/N 9300-042)
96-well, black microplate (Greiner Bio,
FluoTrac 200, 655076)
PN 997-9306
•
Affinity-purified recombinant Aequorea
coerulescens green fluorescent protein
(rAcGFP1, Catalog No. 632502).
Note: rAcGFP1 should be stored at
–20°C.
Other Materials:
· Adjustable p200 Volume Pipettor and Tips
· Adjustable p20 Volume Pipettor and Tips
· TE Buffer (10 mM Tris-HCl, 1 mM EDTA,
pH 8.0)
· 1.5 mL microfuge tubes
· Test tube rack
· Nitrile, vinyl, or latex gloves
EXPERIMENTAL PROTOCOL
1. Instrument Set Up
• Insert the Blue Fluorescence Optical Kit
(excitation 460 nm, emission 515 - 580
nm) into the Modulus™ Microplate
according to the Operating Manual.
• From the Home screen, touch Select
Protocol and follow the wizard. Select
preset protocol for rAcGFP1. Select
fluorescence and the Preset tab for
rAcGFP1; Finish.
• The Instrument Control screen shows
the reading parameters: 1 sec.
integration, Blue Optical Kit, and all
plate wells are selected.
Note: If necessary, use the Plate/Well
selection icon to select or deselect wells
to read. Green is selected and gray is
deselected.
• Refer to the on-screen Help topics,
Quick Start Guide, or Operating Manual
for detailed instructions.
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•
•
2. Preparing Standard Curve
•
Remove the plate after measurement.
Plot RFU values versus dilution
concentrations to obtain a standard
curve.
Prepare dilutions of rAcGFP1 according
to the table below.
µL of
Stock
rAcGFP1
(100
ng/µL)
µL of
TE
Buffer
Final Conc.
(ng/µL)
1
—
—
100
0
2
1
—
99
1.00E+05
y = 9.804x
2
R = 1.000
RFU
Sample #
rAcGFP1 Linearity
µL of
Stock
rAcGFP1
(1 ng/µL)
1.00E+03
0.01
3
5
—
95
0.05
4
10
—
90
0.1
5
25
—
75
0.25
6
50
—
50
0.5
7
100
—
0
8
—
2.5
97.5
9
—
5
95
5
10
—
10
90
10
1.00E+01
1.00E+00
1.00E+02
1.00E+04
rAcGFP1 Concentration (pg/µL)
Figure 1. Concentration of rAcGFP1 in pg/µLversus
RFU. Various dilutions of rAcGFP1 plotted against RFU
values. The Modulus™ Microplate Fluorometer is able
to detect as low as 10 pg/µL and up to 10 ng/µL of
rAcGFP1.
1
2.5
•
Use the standard curve to determine
each samples’ unknown concentration.
Table 1. Preparation of rAcGFP1 dilutions. To make a
range of rAcGFP1 dilutions, add appropriate volumes of
stock rAcGFP1 and TE Buffer as shown in the table.
•
•
•
•
•
Add 200 µL of sample to each well.
Note: If necessary, use the Plate/Well
selection icon to select or deselect wells
to read. Green is selected and gray is
deselected.
Open the instrument door by using the
Door icon on the touch screen. Place
well A1 at the top right corner of the
microplate sample tray. Close the door
by using the Door icon.
Select the Start icon on the touch
screen to begin reading.
The Results screen displays RFU
values immediately after each well is
measured.
Once all measurements are complete,
data can be transferred to an external
computer for further data analysis in
Excel by using the provided USB flash
drive.
PN 997-9306
RESULTS
Sensitivity: < 10 pg/µL
Dynamic Range: Dynamic range of up to
four orders of magnitude
Minimum Detection Limit: 0.28 pg/µL (as
calculated using three times standard
deviation of the assay background)
CONCLUSION
The Modulus™ Microplate Fluorometer
offers superior sensitivity and dynamic range
for detection of Clontech Living Colors®
rAcGFP1, enhancing the results of gene
expression and real-time protein
visualization studies. The Modulus™
Microplate Fluorometer achieves its superior
performance with a dedicated fluorescence
detector instead of sharing the detector with
other detection modes. The individual
optical kit with solid-state optics and a
powerful wavelength-matched LED delivers
excellent sensitivity and dynamic range.
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The modular approach of the Modulus™
Microplate Fluorometer allows for instrument
capability expansion as needs in the lab
change. Luminescence and/or absorbance
detection modules as well as other
accessories can be added after the initial
purchase.
The superior performance, ease of use, and
utmost flexibility of the Modulus™ Microplate
make it an ideal microplate reader for
today’s life science laboratory.
TRADEMARKS
Modulus™ is a trademark of Turner
BioSystems, Inc. Living Colors® is a
registered trademark of Clontech
Laboratories, Inc.
TURNER BIOSYSTEMS CONTACT
INFORMATION:
Orders for Turner BioSystems’ products may
be placed by:
Phone: (408) 636-2400 or
Toll Free: (888) 636-2401 (US and Canada)
Fax:
(408) 737-7919
Web Site: www.turnerbiosystems.com
E-Mail: sales@turnerbiosystems.com
Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085 USA
PN 997-9306
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