Fluorescence Imaging Apllications Guide Addendum

Fluorescence Imaging Applications Guide
Addendum
Since publication of the original guide, a new multi-colour 3. Sensitivity
fluorescence scanning system has been introduced. The
Typhoon 8600 has two excitation sources (NdYAG and The “Normal” sensitivity setting is used for most
HeNe lasers with excitation wavelengths of 532 nm and fluorescent scans. Selecting the medium or high sensitivity
633 nm) and two photo-multiplier tubes (PMTs) that allow settings in Scanner Control may increase the signal-to-
for simultaneous scanning of multiple fluorophores. This noise ratio by acquiring additional signal from the sample.
addendum outlines the recommended instrument setup and Compared to the normal setting, the medium and high
expected performance for the Typhoon. sensitivity settings will result in increased scan time but
will not affect file size.
Typhoon operations 4. Pixel size
1. Filter 200 micron is the lowest resolution setting and is sufficient

for most samples. If higher resolution is required, select a
Six standard fluorescence emission filters are available with smaller pixel size (100 micron or 50 micron) in Scanner
Typhoon. The user selects filters, through Scanner Control – this will increase both the scan time and the file
Control software, that are appropriate for the size.
fluorochrome labels in their experiment. Although
Typhoon has a general laser blocking filter, for best results, 5. Calibration
we recommend using a selected emission filter for each
fluorescent scan. Calibration is not required for Typhoon 8600.
Two different beamsplitters are also available for use in

simultaneous two-channel fluorescent scans. The
combined fluorescent signal from the sample is separated
and directed by the beamsplitter to the two selected
emission filters and then to the two PMTs.
2. Photomultiplier Tube (PMT)
The PMT voltage recommended in each case is a good

starting point from which further optimization may be
needed. The optimum voltage will differ for each
experiment, based on type and concentration of
fluorophore, sample media (i.e., agarose, polyacrylamide)
and thickness. For quantitative results, always use
ImageQuant software (refer to the ImageQuant software
manual) to check for saturated pixels in the image. If pixel
saturation occurs, rescan at a lower PMT voltage setting.
Ethidium Bromide
Instrument setup
Excitation 532 nm
Emission filter 610BP30
PMT voltage 400 to 600
Detection limits
Agarose gel 100 pg/band DNA markers in agarose gel stained with ethidium bromide and
imaged with Typhoon.
Linear range of detection
Typhoon ~100-fold
SYBR™ Gold
Instrument setup
Excitation 532 nm
Emission filter 526SP
Detection limits
Agarose gel 25 pg/band
DNA markers in polyacrylamide gel stained with SYBR Gold and
Polyacrylamide gel 10 pg/band

Typhoon ~100-fold
SYBR Green I
Instrument setup
Excitation 532 nm
Detection limits PCR products in agarose gel stained with SYBR Green I and imaged
Agarose gel 25 pg/band with Typhoon.
Polyacrylamide gel 10 pg/band

Typhoon ~100-fold
Vistra™ Green
Instrument setup
Excitation 532 nm
Detection limits
Agarose gel 25 pg/band
Polyacrylamide gel 10 pg/band DNA markers in agarose gel stained with Vistra Green and imaged
with Typhoon.
Typhoon ~100-fold
Fluorescence 5’
Oligolabelling Kit
Instrument setup
Excitation 532 nm
Detection limits
Polyacrylamide gel 1 fmol/band*
*End-labelled DNA oligo

Typhoon ~200-fold in polyacrylamide gel
Pico Green™
Instrument setup
Excitation 532 nm
Lambda DNA solution in microplate stained with Pico Green and
Detection limits
10 ng/ml

Typhoon ~500-fold
ECF™ Random Prime
Labelling and Signal
Amplification Kit
Instrument setup
Excitation 532 nm
Detection limits (ECF only)

Southern blot 0.25 pg target in 0.5 µg human
genomic DNA

Typhoon ~50-fold
ECF Substrate or AttoPhos™

Instrument setup
Excitation 532 nm
Emission filter 526SP Tubulin slot blot on PVDF membrane detected with ECF substrate
PMT voltage 400 to 600 and imaged with Typhoon.
Detection limits
Western blot 1 ng purified tubulin
genomic DNA

Typhoon ~50-fold
DDAO Phosphate
Instrument setup
Excitation 633 nm
Emission filter 670BP30 Tubulin Western detected with DDAO Phosphate substrate and
PMT voltage 400 to 600 imaged with Typhoon.
Detection limits
genomic DNA

Typhoon ~50-fold
SYPRO™ Orange
Instrument setup
Excitation 532 nm
Detection limits
Polyacrylamide 2 ng/band
Bovine serum albumin protein in SDS-PAGE stained with SYPRO
Linear range of detection Orange and imaged with Typhoon.
Typhoon ~500-fold
SYPRO Red
Instrument setup
Excitation 532 nm
Detection limits
Polyacrylamide 2 ng/band Bovine serum albumin protein in SDS-PAGE stained with SYPRO
Red and imaged with Typhoon.
Typhoon ~200-fold
NEW!
SYPRO Ruby
Benefits
© Ready to use
© One-step staining protocol
© As sensitive as silver staining
© Compatible with mass spec, microsequencing
Uses
Protein detection, especially in 2D gels
Excitation maximum
280, 450 nm
Proteins resolved in a 2D gel, stained with SYPRO Ruby and
Emission maximum imaged with Typhoon.
610 nm
Method
© Run gel.
© For 2D gels, fix for 30 minutes in 10% methanol and
7% acetic acid.
© Add a sufficient volume of undiluted SYPRO Ruby
stain to an appropriate polypropylene container
(enough to cover the gel).
© Place gel in stain and cover container with foil.
© Incubate with orbital shaking (50 rpm) for 90 minutes
to 3 hours.
© (Optional) To decrease background fluorescence,
rinse gel in either deionized water or 10% methanol
and 7% acetic acid for 30 minutes
Instrument setup
Excitation 532 nm
Detection limits
Polyacrylamide 4 ng/band

Typhoon ~500-fold
ECF Western Blotting Kit
Instrument setup
Excitation 532 nm
Detection limits (ECF only)


Typhoon ~50-fold
ECF Western Blotting

Reagent Pack
Instrument setup
Excitation 532 nm
Detection limits
Tubulin western detected with ECF Western Reagents and imaged
Linear range of detection with Typhoon.
Typhoon ~50-fold
Fluorescein
Instrument setup
Excitation 532 nm
Detection limits Fluorescein-labelled PCR products imaged with Typhoon.

Polyacrylamide gel 400 amol/band*

Cy™5
Instrument setup
Excitation 633 nm
Emission Filter 670BP30
Differential display reactions using Cy5 label and imaged with
PMT Voltage 400 to 600 Typhoon.
Detection limits

Cy3
Instrument setup
Excitation 532 nm
Direct fluorescent Cy5 Western blot imaged with Typhoon.
Detection limits

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Fluorescence Imaging Applications Guide

Typhoon operations 4. Pixel size

1. Filter 200 micron is the lowest resolution setting and is sufficient

Two different beamsplitters are also available for use in

2. Photomultiplier Tube (PMT)

The PMT voltage recommended in each case is a good

Linear range of detection

Linear range of detection

Linear range of detection

Linear range of detection

Detection limits (ECF only)

Linear range of detection

ECF Substrate or AttoPhos™

Linear range of detection

Linear range of detection

Linear range of detection

Detection limits (ECF only)

Linear range of detection

ECF Western Blotting

Detection limits Fluorescein-labelled PCR products imaged with Typhoon.

Linear range of detection

Linear range of detection

Linear range of detection

Amersham Pharmacia Biotech UK Limited Typhoon , ImageQuant , Vistra , Cy , ECF , and

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