Molecular Dynamics Phospho-Imager
Guidelines
Logging In
Press Control ­ Alt ­ Delete to
bring up log in screen
Type in username and password (Same
log-in as you would use on bioinformatics computers)
Microsoft
excel will open on startup
Scanning (Using Phosphor-screen)
Check to see if the phospho-imager
is on. There should be a green light on in the upper left hand corner of the
machine.
Open up the lid and place your
screen on the scanner.
Open up the Storm Scanner Control
program located on the desktop
Check to make sure all the
parameters are what you want
Phosphoimager
vs Fluorescence
Resolution/Pixel
Size (200 microns is normal)
Scanning
Area ­ select the smallest area necessary to make your scan
Click on Scan
It will bring up a box to save the
file
Create
a folder for the lab on the F drive
Make
sure to change the file name so that it will not overwrite an old scan
Click
on Save
After it is finished scanning, you
can quit the program.
Scanning (Westerns)
After treating membrane as you
would normally do with ECL+, place the membrane on a glass plate, face up.
It is important to note that you NEED to use ECL+ and not regular ECL if you want to
detect your bands with the Phosphor Imager
Wrap the glass/membrane with Seal
View sealing film (not regular saran wrap because you will get clouding).
7/04 ­ An alternative to the Seal
View is to use a Write-On Transprancy. These are supposed to work very well.
Place the glass/membrane face down
in the phosphoimager.
De-click Red Flouresence so that
only Blue flouresence is chosen.
Choose area
Scan
Image Analysis
Open
up the Image Quant program located on the desktop
Under the file menu, select open
and locate your scanned image in the lab folder on the F Drive
Choose the oval tool to mark a
specific area to be analyzed.
Drag and click a small region that
surrounds the band to be analyzed.
Click on the circle after you have
completed the first one, and Copy and Paste it, so that every subsequent
region is identical. You can move the circles by dragging them, or using the
arrow keys to cover the next region to be quantified.
The circles will be numbered
sequentially for analysis.
Remember to place a final circle
outside of the gel region so that you can produce a background band.
All of the bands that you would like to
quantify are encircled, go to the Analysis pull-down menu, and select volume
report.
A readout will appear. In order to
save it, you have to save it in an Excel format. (It will ask you if you want
to do this, so click yes.)
Now you can do your own analysis.