ImageLab software version 4. 1 Image acquisition and densitometric analysis. For background (called rolling disc in the software) subtraction, a value of 1 Fig. In the end, the integrity and robustness of the published scientific data should be part of the considerations while planning image analysis experiments. select and determine the background-subtracted density of the bands in all the gels and blots (Fig. You should be able to apply brightness, contrast, and color balance adjustments without altering your data. Sometimes, a qualitative interpretation might be better suitable than a flawed quantification. When analyzing Western blots, look for a software that does not change the raw data and adjusts the image display uniformly. IMHO: Not because we can measure everything means also we should do it. I can only recommend to use either the software of the Western Blot imaging system in the lab or alternatively GelAnalyzer because it allows to stick pretty well to the recommended procedure by Western Blot material suppliers (which I would guess are the most experienced people in that particular field) and as seen in the publication above. Hammond, “A defined methodology for reliable quantification of Western blot data.,” Mol. So, generally there are many pitfalls related to WB measurements.īesides the literature list in one of the linked posts above, mainly the following gives a good insight into the procedure: The quantification will reflect the relative amounts as a ratio of each protein band relative to the lane’s loading control. And band selections which overshoot the actual band also lead to wrong results. Quantifications of Western Blots with ImageJ by Hossein Davarinejad This protocol will allow you to relatively (no absolute values) quantify protein bands from western blot films. One cannot reliably quantify bands by making boxes around them and using analyze measure nor do horizontal lines fulfill the requirements. Here is one video which is explaining a little more detailed the considerations of WB in general, normalization as well as measurements The latter and pretty much of most other ones completely ignore every thing mentioned in scientific literature regarding Western Blot measurements (and the pre-requisites for it). There are many videos online like the linked one above. But I cannot contain myself to add my 2 cents to this topic, because some of those videos make me like… No offense to no one making those videos or taking them as orientation in case of the lack of other available resources. This was the one I looked at Analysing blots and gels with ImageJ/Fiji - YouTube
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