Download Determination of Fluorescent Proteins

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Application Information
Determination of Fluorescent Proteins
(eGFP, YFP, CFP and DsRed) Using
the Cell Lab Quanta™ SC MPL
Yong Song MD, PhD
Cellular Analysis Business Center, Beckman Coulter, Inc., Miami, FL 33196
INTRODUCTION
Green fluorescent protein (GFP) is a biological
detection molecule that was discovered in the natural
world. GFP is derived from the jellyfish Aequorea
victoria which contains a sophisticated two-stage,
light-generating system using a bioluminescent
protein, Aequorin. In the presence of calcium, the
Aequorin protein will emit blue light. This blue light
will then excite a 28kDa green fluorescent protein
(GFP), subsequently causing the jellyfish to appear
green. If the GFP protein is absent, the jellyfish will
emit blue luminescence at an emission maximum at
466 nm.(1)
GFP contains a chromophore which absorbs
blue light and allows the emission of green light. This
chromophore is biosynthetically created between
amino acid residues 65-67 (Ser-Tyr-Gly) of the GFP
protein.
By using the appropriate transcription vector,
researchers have been able to add the DNA
sequence of their protein of interest to the GFP
DNA sequence. When this chimeric construct is
introduced into a cell, the host cell will express a
fusion protein containing GFP and the protein of
interest or co-express GFP and the protein of
interest. Thus, the expression level of the transfected
protein can be measured by determining the
fluorescent intensity of GFP in the cells. Uniquely,
this approach allows cells to be studied in vivo
without having to be fixed or stained. GFP has been
used as a cell marker, reporter gene and fusion tag.
GFP has also been used as an active indicator for
many protein functional studies such as protease
action, transcription factor dimerization, calcium
sensitivity and quantitative measurement of gene
expression.(2)
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Various forms of GFP, such as enhanced (eGFP),
have been created using basic molecular biology
techniques to alter the DNA sequence and the
critical amino acids that make up the chromophore
within the GFP molecule. Many other subtypes of
fluorescent proteins such as cyan (CFP), yellow
(YFP) and red (such as DsRed) have been created
and used as probes to proteins expressed in live
cells.(3)
The Cell Lab Quanta™ SC is a flow cytometry
system designed to simultaneously measure
Electronic Cell Volume (EV), side scatter and 3-color
fluorescence. With the optional multi-plate loader
(MPL), the Quanta SC MPL is capable of handling
24, 96 or 384-well plates and microcentrifuge tubes
for much higher throughput analysis.
In this Application Information bulletin, examples
of the determination of eGFP, CFP, YFP and DsRed
expression using the Quanta SC MPL are described.
EGFP has excitation and emission maxima
at 489 nm and 509 nm, respectively (Figure 1).
YFP’s peak excitation and emission wavelengths
Figure 1: Excitation/emission spectra of GFP.
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are 513 nm and 527 nm. Both eGFP and YFP can
be excited with a 488 nm blue laser.
CFP has a peak excitation wavelength at
434 nm. It can be excited with a UV mercury arc
lamp using a 425/40 nm BP excitation filter. Its
peak emission is at 477 nm.
Although DsRed’s peak excitation is 556 nm, it
has a very broad excitation spectrum and can be
excited with a 488 nm laser. Its peak emission is at
586 nm.
For GFP (FL1), YFP (FL1), CFP (FL1) or DsRed
(FL3) measurements, select the “Log” display under
“Parameter Info” and adjust the FL1 or FL3 PMT
voltages to locate the populations of control or
fluorescent protein transfected cells completely on
scale. The FL1 or FL3 negative cells should be on
the edge of the first log decade population and can
be confirmed with control cells. Occasionally
enhanced GFP and YFP containing cells are very
bright. In some cases, it is recommended to use less
laser power so that the brightest cells can also be
displayed on scale. The Quanta SC’s 488 nm laser
output can be adjusted from 2-22 mW.
MATERIALS
CC Standard L10 Polysterene Latex, Beckman
Coulter PN 6602796
FILTER CONFIGURATION
EGFP, YFP and DsRed
Phosphate Buffered Saline (PBS)
Control Cells (control vector trasfected or
untransfected cells)
The standard laser 488 nm laser filter configuration
is used for eGFP (FL1), YFP (FL1), and DsRed
(FL3) measurements. DsRed can also be detected
on FL2.
Transfected Cells
SAMPLE PREPARATION
• Excitation: 488 nm Laser
• Emission
– 525/40 BP (FL1)
– 575/30 BP (FL2)
– 670LP (FL3)
Prepare the control and transfected cells in PBS to
a concentration of 0.1-2 x 106 cells/mL according
to the established laboratory procedure.
ACQUISITION PROTOCOL SETUP ON
THE QUANTA SC MPL COLLECTION
SOFTWARE
CFP
For CFP measurement, the filter configuration has to
be modified (Figure 3).
The Electronic Volume (EV) is used to accurately
measure cell size and can be calibrated using CC
Standard L10 Polystyrene Latex. Once the size scale
has been calibrated, the entire cell population can be
brought on scale by adjusting the EV gain. Debris
can be gated out by adjusting the Lower Level
Discriminator (LLD). Additionally establish a polygon
region on the cell population using EV/SS dual
parameter display (Figure 2).
• Excitation: Mercury arc lamp with a 425/40 nm
BP excitation filter. This can be obtained from
various optical filter providers such as Chroma
Technology Corp. (Part: 50 mm D425/50mblocked).
• Emission
– 480/40 BP (FL1)
(This filter can also be obtained from Chroma
Technology Corp. Part: 25 mm, ET480/40m)
Figure 3: Filter Block Configuration for CFP detection.
Figure 2: Example of establishing a polygon region to gate
out debris.
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(B)
(A)
Control
Control
GFP+ 94%
YFP+ 48%
GFP
YFP
(D)
(C)
Control
Control
CFP+ 98%
DsRed+ 45%
CFP
DsRed
Figure 4: Overlaying histograms of control empty vector transfected (B, D, red) or untransfected (A, C, red) cells comparing to stably
(A, C) or transiently (B, D) transfected cells that expressed GFP (A, green), YFP (B, blue), CFP (C, blue) or DsRed (D, blue).
SELECTED REFERENCES
1. Prasher DD, Eckenrode VK, Ward WW,
Prendergast FG, Cormier MJ 1992. Primary
Structure of the Aequorea Victoria greenfluorescent protein. Gene. 111, 229 – 233.
2. Tsien RY 1998. The Green Fluorescent Protein.
Annual Review of Biochemistry. 67, 509 – 544.
3. Shaner NC, Steinbach PA, Tsien RY 2005. A
Guide to choosing Nature Method, 2, 905-909.
3
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