(Dept Cell Biology - NYU)
NB= Phosphorimager cassettes cost` $2000. They must be treated with respect.
Always store them flat at all times.
Do not use Tape.
Do not use "Enhance"
NB = Try to place the blots in the cassette horizontally and try to limit the space used
- Open the cover of the apparatus
- Place the screen of the cassette face screen down
NB = the top label of the cover of the cassette is orientated on the left
NB = for little screens, use the black mask
NB = never touch the white part of the screen
- Secure with red caps
- Close the cover of the apparatus
On the computer:
- "Enter" ---> menu
- "A) Phosphorimager" ---> "Enter"
- Enter username ---> Cowinp01
- Enter password ---> ipswich
- "Phosphorimager" ---> "Enter"
- Go into "Scanner" ---> "Scan sample"
Enter "From a2"
Enter "To l13"
Enter "File name"
Pixel size is 176U ---> possible to change to 88U
- The image is obtained ---> change the size of the square to obtain the entire image of the blot
- Go into "View" ---> "Grey color adjust"
Play by changing the values
Hi (500.00, 100.00)
Lo (0.5, 1.00, 2.00)
Play with the curve to modify the background
Do "Set range"
- Go to "File" ---> "Printer set-up"
If the image is too big, change "portrait" to "landscape"
- Go to "File" ---> "Print"
SET TO 1X
- To export the image
In the file, put the square in the right bottom part of the image of the blot
Note the values X2 and Y2
Go into "Export image"
Enter the values X1 = 0, X2, Y1 = 0, Y2
Save file as "manue1.tif"
Close all the files
Go back to menu ---> "C) File transfer to SPARC"
Enter "manue1.tif" 2X
Fix the zip drive and set up the disk drive on the computer
Go back to menu ---> "B) " ---> Enter
Answer by entering the filename and "yes"
Erase the screen by putting the screen face screen on the light box for 12 min.
To quantify signal
Objects = left -part of mouse ---> rectangle (or ellipse for less background)
Draw a square with the left part of mouse on what you want to quantify
Take background defined around the rectangle
Rectangle numbered 00001
Integrate the number of picksets and densities
Mean value of intensity around the frame but integrated on all the area
Integrate as a value of background
total - background = value of the band
Part left of mouse Quan ---> integrate volume (and not integrate area)
--->Select with left part of mouse until the end
Background values = use local background
Background done = mean background at the level of the line
Select columns before to print by "blacking"