I am wondering the implications of imaging western blots that have been vertically inverted (i.e. lanes still run 1-15 from left to right, but columns run from lightest to heaviest from top to bottom, respectively). Gels transferred proteins onto nitrocellulose membranes, and primary/secondary antibody incubations were good. I know this because signal strength seemed strong with all targets, but I had to carefully consider molecular weights when target probes yielded >1 band of interest in proximal molecular weights.
I have been unable to find a systematic or sufficient answer as to if vertical inversion of blots during imaging would confound signal strength results after conducting densitometric analysis in ImageJ. A subsequent western using same samples yielded somewhat different results but sample size and strength of effects were small/non-significant.
I appreciate insight/advice on this matter.
Why do you think that image-based densitometry should depend on the orientation of the blot? What would be the physical principle behind this?
Anyhow, a simple fix is just to flip the image in image J (Image -> Transform -> Flip Vertically).
Without a full understanding, I recognize that the proteins transferred more directly onto the side of the blot that was not imaged. While there was clear binding of primaries and secondaries to targets (as they were ECL-reacted), I do not know if signal strength would be different if I had imaged the other side of the blot; I suppose the physical principle behind this would concern protein migration distance from gel to membrane and consideration of protein abundance within the membrane itself.
I conducted the densitometric analysis using the vertical-invert function but want to make certain the signal strength is not different/counfounded if I imaged the blots on the side that more directly received the proteins during transfer.
Oh I see. The blot itself was imaged upside down. Sorry for the misunderstanding.
I suppose the same considerations would apply if you were to image the blot right side up -- i.e. some of the proteins could theoretically migrate deeper into the membrane then others based on molecular weight. However, I would think that should not be important differences when comparing bands in different lanes representing the same protein. It could potentially introduce differences if you are normalizing one protein's abundance by another's.
- Badges
- Science method