In the end, the integrity and robustness of the published scientific data should be part of the considerations while planning image analysis experiments. ![]() Rehbein & Schwalbe, 2015), and the peak maximum intensity has been utilized in the quantification of proteins in Western blots imaged by fluorescence (Gürtler et al., 2013 Holzmüller & Kulozik, 2016). Sometimes, a qualitative interpretation might be better suitable than a flawed quantification. Images of the SDSPAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. 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: 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. In order to achieve quantitative densitometric data from western blots, determination of the appropriate total protein load assures optimal dose-dependent responses per target (Figs. 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). Quantifying the intensity of the bands on a Western blot can provide valuable information about protein expression levels. Image software allows for accurate and efficient quantification of Western blots by measuring the intensity of the bands. There are many videos online like the linked one above. To begin the quantification process, the first step is to upload the image into the image software. 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 For quantification of the entire volume of a Z-stack, it is recommended to use more advanced image analysis programs (such as Imaris, Volocity, etc.) instead of ImageJ/FIJI as these programs enable 3D segmentation.
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