Detection of Proteins
(Amersham ECL Plus Western Blotting Detection Reagents; GE Healthcare #RPN 2132)
1. Remove the detection reagents from storage at 4°C and allow them to equilibrate at room
temperature.
2. Mix detection solutions A and B in a ratio of 40:1 (5 ml solution A + 125 l solution B)
3. Drain excess wash buffer from the washed membranes and place protein side up on a
sheet of Saran wrap or reaction folders. Pipette the mixed detection reagent onto the
membrane.
4. Reagents should cover the entire surface of the membrane (they are held by surface
tension onto the surface of the membrane)
5. Incubate for 5 min at RT.
Chemifluorescent detection
1. Drain off excess of detection reagent by reclining gently the membrane and touching the
edge against a tissue.
2. Place the blot (protein side up) on the scanning bed of the BioRad Chemidoc MP.
a. Launch the Image lab software.
b. In the “protocols” window click on the “Open…” button.
c. Go to: desktop/Schipani/Protocols and select WB-ECL plus 1-60 s
The settings of this protocol are:
- Application: Chemi Hi sensitivity
- Size: Bio Rad Criterion gel
- Exposure: signal accumulation (10 images from 1 to 60 s of
exposure) with highlight of saturated pixels.
- Color: gray.
d. Click on “position gel” and center your membrane
e. Click on “run protocol”
f. After imaging sequence, right click on one of the miniature and select “save all”
g. Choose the appropriate destination folder: desktop/Schipani/month-year and click
“select folder”.
h. Finally save your data on a storage device or send them to yourself by email for
quantification.
Updated 03/10/14 by CM
Chemiluminescent detection
1. Drain off excess reagent by holding membrane gently with forceps and touching the edge
against a Kimwipe
2. Place the blots, protein side up, on a fresh reaction folder inside an x-ray film cassette,
close the folder and gently smooth out any air bubbles. Place a glow in the dark sticker on
top of the reaction folder on the left side of each membrane, making sure it does not
cover the membranes.
 Avoid putting too much pressure on the membrane
 Ensure that there is no free detection reagent on the outer side of the reaction
folder; the film must not get wet
3. IN THE DARK ROOM, place a sheet of autoradiography film on top of the membrane.
Close the cassette and expose. The first exposure time is variable and depends on the
strength of the signal. Approximately a good signal should be visible after 15 seconds.
4. Remove the film and replace with a second sheet of unexposed film
5. Develop the first piece immediately on basis of its appearance, estimate how long to
continue the exposure of the second piece of film. Second exposures can vary from 1 min
to 1 hr.
Updated 03/10/14 by CM