Q. What is a Typhoon system?
A. The Typhoon 8600 system is a versatile multimode scanner with capabilities for storage phosphor, fluorescence, and chemiluminescence detection of gels. The instrument combines powerful laser excitation sources with efficient optics for sensitive fluorescent imaging. Emissions are recorded in an image file for quantitative analysis.
Q. Will the Typhoon system do ethidium bromide?
A. Yes. Molecular Dynamics do not currently provide sensitivity data for the Typhoon, however the Fluoroimager system is about 5 times more sensitive for ethidium bromide visualization than the traditional UV light box and Polaroid film setup. VISTRA Green, a DNA dye available fromAmersham Pharmacia Biotech, is used the same way as ethidium bromide and offers 100 times greater sensitivity than the traditional ethidium bromide method. See Molecular Dynamics' Application note 64 - Fluorescent DNA Gel Stain Detection for further information and a protocol.
Q. Will the Typhoon system do chemiluminescence?
A. Yes. For high-sensitivity DNA and protein detection on blots, Molecular Dynamics recommends chemifluorescence. Benefits include direct quantitation, no film exposures, and increasing signal strength for up to 12 hours. Chemifluorescent samples can be scanned up to two weeks following sample preparation with no significant loss in signal or resolution. Chemifluorescent reagent kits for Southern, northern and western blotting are available from Amersham Pharmacia Biotech. For more information and a thorough comparison of chemiluminescence and chemifluorescence, see Molecular Dynamics' publication "Understanding Chemifluorescence."
Q. What applications have been characterized for the Typhoon system?
A. Molecular Dynamics' publication "Fluorescence ImagingApplications Guide" has detailed applications information and protocols for DNA and protein detection on blots, gels, and microplates using the Fluoroimager system. Most of this is also applicable to the Typhoon.
Q. How sensitive is the Typhoon system?
A. Molecular Dynamics do not currently provide sensitivity data. Considering the Fluoroimager system, one femtomole of a DNA 24-mer, end-labeled with fluorescein in a 1mm thick 10% acrylamide gel, scanned in high sensitivity mode produces a signal three times greater than background. Smaller quantities are visible on the scanned image but do not always meet the three-times-background detection criterion. Intercalating dyes such as ethidium bromide and Vistra Green typically offer lower limits of detection than those that are achieved with fluorescein end-labeling because there are more dye molecules associated with each DNA molecule.
Q. What is the difference between the "Normal Sensitivity" and "High Sensitivity" scan modes?
A. Selecting the "High Sensitivity" mode causes the Typhoon to scan the sample several times and then average the scan data to reduce background noise on the image. The effect is to reduce the lower limit of detection. Because it takes longer, Molecular Dynamics recommends using the high sensitivity mode only in the event that no bands are visible after a normal mode scan.
Q. Can the Typhoon system detect PCR reactions run directly in microplates?
A. Yes. The Typhoon system requires flat-bottomed microplates. Plates are placed directly on the glass platen for imaging and quantitation
Q. What is the effect of gelthickness on sensitivity?
A. Thinner gels work better. Thick gels produce higher background levels which can lead to a reduction in signal to noise and poorer detection limits.
Q. Where did this FAQ come from?
A. The first 8 questions in this FAQ were modified from Molecular Dynamics' Fluoroimager FAQ.
Q. How does the Typhoon detect fluorescein and similar probes?
A. Probably magically. The physical mechanism of fluorescence dictates that emission must always occur at a longer wavelength than excitation. The Typhoon excites fluorescein or Vistra Green at 532 nm and measures emission at less than 526 nm. We are currently researching this contradiction!
Q. What about detection of proteins in gels?
A. Fluorescent protein gel stains combine high sensitivity with ease of use. The SYPRO Orange and SYPRO Red protein gel stains are one-step fluorescent stains that provide sensitivity that is equivalent to silver staining. These fluorescent stains can be used with both denaturing and native gels and do not interfere with western detection or microsequencing. In addition, the presence of nucleic acids and lipopolysaccharides does not interfere with the ability to detect proteins using the SYPRO stains. The SYPRO Orange and Red stains are optimal for rapid and efficient fluorescent staining of 1-D protein gels. The SYPRO Ruby protein gel stain provides sensitive fluorescent detection of both 1-D and 2-D protein gels. In addition, the SYPRO Ruby stain is compatible with subsequent mass spectrometry and Edman-based sequencing. The Typhoon 8600 instrument provides sensitive fluorescent detection of the SYPRO Red, SYPRO Orange, and SYPRO Ruby protein gel stains. (Consult Molecular Dynamics' Application note 66-Fluorescent Protein Gel Stains for further information and a protocol).
Q. What probes will work with the Typhoon system?
A. Acomprehensive list of fluorescent dyes that should work with the Typhoon system appears in Molecular Dynamics' Fluorescent Imaging Application Guide.
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