I've been using Revert700 total protein stain to detect total protein in my western lanes. While this stain is robust & reversible, I have trouble quantifying in Image Studio Lite (LiCor) and FIJI/Image J (NIH). My primary issue is that both IS Lite & FIJI seem to expect nearly perfectly vertical lanes, which I have yet to achieve. The more a lane deviates from "ideal" the more an ROI/Shape picks up adjacent lane signal (or ladder signal). In IS Lite rotation of shapes causes the background selection to change somewhat dramatically, becoming much larger (in the case of "All" background selection) or further from the target signal (in the case of "Top/Bottom" or "Left/Right" background selection). The freehand tool is painfully tedious, but besides that requires "User-defined" background, which LiCor advises against using when global background levels aren't consistent (and I don't believe mine are). Image J doesn't seem to have the initial background problem I encounter with IS, but I've been unable to perform lane analysis after rectangle shape ROIs are rotated to adjust for the lane angles. I get an error message saying this analysis can only be done on "Rectangular selection".
Until now my work-around has been using multiple shapes per lane in Image Studio Lite and adding their signal values together. While this seem like an adequate approach to determine total protein signal, it complicates the use of IS's 'Concentration' feature, which I have found very useful in determining the accuracy of my results. In short, with multiple shapes I have to report multiple "fractions" of the total signal from my Concentration Standard lanes, and hope that IS can figure out the rest. I'm dubious of this approach and would like something that provides a bit more confidence.
I've added images that are representative of my difficulties. Any guidance would be most appreciated (Blue text-Image Studio Lite; Black text-Image J/FIJI).
My western running/transferring conditions:
- Protein amount varies by experiment, but typically no more than 30ul of sample per well in precast Invitrogen 8% Bis-tris gels (10-17 wells)
- Run in room temp 1x MOPS running buffer @ 200v ~ 30min
- Wet transfer in cold (20 C) 1x transfer buffer onto nitrocellulose membrane @ 100v ~80min
- LiCor Revert700 stain, scan, destain
- Block w/ LiCor blocking buffer before incubating w/ primary antibody overnight
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