Chemifluorescent detection of protein on a Western Blot
Last updated: 1/12/12 By: Tim Starr
Overview
Follow this protocol to detect proteins on a membrane that has been incubated with primary and secondary antibodies
Protocol
Currently I am using chemifluorescent substrate from Amersham Pharmacia kit RPN-5783.
Prepare the substrate by mixing 36 mg ECF substrate in 60 ml ECF buffer. This buffer is good for 2 – 4 weeks at 4º C, or aliquot and freeze at – 20º C.
Tightly wrap saran wrap around hard plastic board.
Pipette 24 µl / cm2 of membrane onto saran wrap. 3” x 3.5” membrane = about 1.5 ml of substrate, but I find that you can use less and still get good signal.
Gently lay membrane (dull/protein side down) on top of ECF substrate, make sure there are no bubbles.
Incubate for 5 to 20 minutes.
Pick up membrane and touch edge to paper towel to partially drain excess fluid.
Then lay the membrane (protein side down) on a transparency lying on top of the Storm 840 Scanner.
Storm 840 scanner is in BIPL. At this point it is free to use, just sign up on the BIPL web site. Machine will image both phosphorescence and chemifluorescence from Alkaline phosphate substrate, which absorbs around 400 something and emits at 540 –560.
Note where the blot is located using the letters across the top and the numbers down the side of the scanner.
Open the PhosphoScanner program from the desktop icon.
Choose Blue fluorescence/chemifluorescence
Highlight the grid squares you want to image. (More squares takes more time).
Choose Setup Set pixel size to 200 microns (unless you need higher resolution)
Set PMT voltage. 0 – 1000. 800 for normal fluorescence. I like 925. Choose OK.
Choose Scan Select a folder/filename for the image file.
Use Drive F “Storage”, Data: TStarr: Select “Do not press gel” (unless you want it squashed).
Machine will scan the blot, save the .gel file to the folder you selected and then open Imagequant with the picture.
At this point you can quantify bands, or you can just save as a .tif file and export for later analysis with other software. I don’t know how to quantitate using Imagequant, but Jon can teach it.
Exit the program. “Do not park scan head”
To transfer the file to the Bipl Snap server Generally the SNAP server is already connected to the computer and will show up as “bipl Snap Server” on My Computer. Just drag the files from Storage Drive F: Data: TStarr into the Bipl Snap Server TStarr folder. From your Mac, use connect to server and transfer onto your own computer