PHOSPHORIMAGER

(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

Do "Scan"

- 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

"Convert"

Save file as "manue1.tif"

Close all the files

Exit

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"