Typhoon - HRP Antibody Detection
updated 08/13/04 - Benjamin Costantino
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Membrane Preparation
1) TURN LASER ON NOW! Needs a 10 min. warmup.
2)Use 1:40 (Solution A:Solution B) of ECL detection Kit. 1 - 2ml would be fine for our typical 8cm x 4cm membrane. (mix solutions by inversion)
3) 4 mls of Sol 'A' and 100µl of Sol. 'B'
4) Remove membrane and gently drain off TPBS by touching corner of membrane with Kim-Wipe.
5) Place membrane, protein side up, in separate tray and add ECL solution to cover the entire membrane. Cover tray with foil and incubate for 2min. room temp.
6) Meanwhile, clean glass of Typhoon
7) Place membrane on glass protein side down at ~D-I/1-4 . Make sure there are no marks on the glass.
8) Pour some solution at the corners of the membrane to keep it wet.
Computer Section
1) Bring up SCANNER program.
2) Go to New.
3) Under Acquisition Mode select Fluorescence under the drop down and click Setup.
Under Emission - select 520 BP 40CY2 Blue Fam and Blue (457). (Adjust PMT if too dark, 620 is default)
Click OK to get out of Setup; In original window click SCAN.
Name file and put into appropriate location
The blot will now scan.
Band Quantification
1) Under View - select tool bar and check off Object.
2) Choose the Squaring tool (under the pointer in the new tool bar you opened).
3) Select a square on your gel background to use as Background.
4) Under Analyses - select Background Correction.
5) Select Object, then check off object average and click Set (make sure to set the RECT and not any numbers).
6) Now square off a band and duplicate as necessary.
7) Move band squares into place over bands.
8) Set the band values by selecting Object and checking off object average; click set when completed. ( this procedure is the same thing as 3 lines up)
9) Under Analysis - select Volume Report.
10) Select All and click Report.
11) Double click on read out to get into Excel.
To save image of Western- Under Edit.
