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.