In the end, the integrity and robustness of the published scientific data should be part of the considerations while planning image analysis experiments. Sometimes, a qualitative interpretation might be better suitable than a flawed quantification. IMHO: Not because we can measure everything means also we should do it. Upper panel, Western blot analysis of tubulin contents in mouse livers. (C) Evaluation of the consistency of QDB analysis with Western blot analysis. The quantification will reflect the relative amounts as a ratio of each protein band relative to the lane’s loading control. 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. This image is converted digitally using Image Studio Digits from Li-Cor (Lincoln, NE, USA), and plotted with the result of QDB analysis in the upper panel for comparison purpose. 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.
![using imagej for western blot quantification using imagej for western blot quantification](https://www.frontiersin.org/files/Articles/319969/fimmu-09-01126-HTML-r1/image_m/fimmu-09-01126-g005.jpg)
Hammond, “A defined methodology for reliable quantification of Western blot data.,” Mol. All data were imaged using the Invitrogen iBright FL1000 Imaging System. This technical note provides the basic principles of normalization using internal loading controls and describes how to accurately normalize western blots to obtain meaningful, reproducible data. 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: make normalizing the western blot essential. And band selections which overshoot the actual band also lead to wrong results. 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… How to put rectangles in the image to quantify6. How to rotate image in imagej to make the lanes in the straight line5. How to adjust background/ brightness and contrast of western blot image in imagej4. No offense to no one making those videos or taking them as orientation in case of the lack of other available resources. How to upload western blot image in imagej2. This was the one I looked at Analysing blots and gels with ImageJ/Fiji - YouTube