En charge du Storm : Serge Hardy En charge de l'Odyssey® : en cours
Phospho-imageur Storm 840 (Molecular Dynamics)
The Storm 840 allows the detection and the measure of radioactivity and fluorescence of solid surfaces (gels, membranes,…).
Detection of radioactivity
The storm replaces or comes to supplement the autoradiography. It is more sensitive but also the zone of linearity is wider (105 compare to 102) that allows more reliable quantifications.
The Storm technology is based on the « capture » of radioactivity on a phosphorus storage screen. A laser beam then excites the screen, the resulting emission that is proportional to the quantity of radioactivity initially present in the sample creates an image.
The digitalised image can be analysed for quantification with the program Image Quant (licence for IGDR). This program works only on Windows NT.
To use the Storm, the users must buy their own screens. There are three kind of screen:
- Screens for “general use” to detect high energy radiation emitted by 125I and 32P or medium energy radiation emitted by 33P and 35S
- Screens "low energy" to detect more efficiently medium and low radiation emitted by 33P, 35S and 14C
- Screens "tritium" to detect very low energy emitted by 3H
Fluorescence detection
The STORM 840 is equipped to directly detect (without any screen) blue fluorescence and chemofluorescence (excitation 450 nm & emission 540-560 nm)
To use the STORM 840, one must contact the person in charge of the equipment who will open an user file on the computer. The machine is on free acces for registered users, no booking is required. The hard disk of the computer has a very low memory capacity, consequently it is asked not to store any data on STORM computer. To help users the computer is linked to the network and is equipped with a CD burner.
Imageur infrarouge Odyssey®
The Odyssey® Infrared Imaging system allows direct infrared fluorescence detection by use of fluorophores emitting in infrared wavelengths where auto-fluorescence and light scattering due to plastics or membranes are dramatically reduced. Odyssey® imaging gives then a better signal-to-noise ratio and provides accurate quantification in a wide linear range.
Infrared dyes used for Odyssey imaging (IRDye®, figure above) have an excitation wavelength of either 680 or 780 nm and an emission wavelength of 715 and 815 nm respectively. These dyes can be detected independently and simultaneously, allowing for the normalization of a given protein by another reference protein (Two-Color Western Blot).
Current applications of Odyssey® imaging are:
- Two-Color Western Blot with possibilities of quantification
- In Cell Western Blots (in 96 or 384-well plates)
- Reading of gels stained with Coomassie Blue or SyproRed
- Fluorophore-Linked Immunosorbent Assay (FLISA) : alternative to ELISA
Besides classical material necessary for each application, you will only need IRDye® secondary antibodies specific for detection by Odyssey Imaging. Antibodies raised against mouse (IRDye®680) and against rabbit (IRDye®780) are available in IGDR. You can also order antibodies raised against other species or Odyssey® Blocking Buffer at Li-Cor Biosciences.
