This video is about western blot analysis with ImageJ, quantifying bands on SDS-PAGE using ImageJ, ImageJ tutorial, Using ImageJ to quantify proteins bands, western blot densitometry in ImageJ, quantifying intensity in imagej, imagej quantify gel bands, western blotting, western blot data, blot data, western data, or Immunoblotting. Hopefully it helps if you're new to the topic/technique. This channel takes you through some of the techniques and concepts I've learnt working as a Research Assistant. I’ve worked in medical research for years and want to be useful to people new to the lab life. I'm Adwoa (Adwoa Biotech), a Biotechnology graduate. PREVIOUS VIDEO USING INTEGRATED INTENSITY IN IMAGEJ: As each peak is selected, a value is given for that peak.Ĭopy the values and analyse in excel or another spreadsheet program as shown in the previous video. use the ‘wand’ tool to select each of the peak. use the ‘line’ tool to mark the base of each peakĨ. This shows you individual peaks associated with each protein band in the lane.ħ. Move the rectangle to all additional lanes and SELECT NEXT LANE after each move to register the lane go back to ANALYZE - GELS - SELECT NEXT LANE Use the forward arrow of your keyboard to move to the next lane.ĥ. Go to ANALYZE - GELS - SELECT FIRST LANEĤ. draw around the lane you wish to quantifyģ. The method is also preferred when the bands you need to quantify are very faint.Ģ. That is, different proteins that cannot be quantified together as an Integrated Density. This method is useful when you have multiple western blot bands that are distinct from each other. Multiplexing can capture up to four proteins in a single blot for more meaningful and representative experiments, a must for western blot publication. Fluorescence detection multiplexing achieves more data from your sample with iBright. In this video I show the NIH-recommend process for quantifying bands derived from SDS-PAGE.īelow are the instructions for using the area under the peak method in your western blot quantitation. 2023 Guide to Quantitative Western Blot Publication. ImageJ is a public domain program developed by Wayne Rasband while at the National Institutes of Health (NIH). In the end, the integrity and robustness of the published scientific data should be part of the considerations while planning image analysis experiments.Subscribe for a fun approach to learning lab techniques: 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. 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:
![quantifications of western blots with imagej quantifications of western blots with imagej](https://media.springernature.com/lw685/springer-static/image/art%3A10.1007%2Fs12035-024-04054-2/MediaObjects/12035_2024_4054_Fig2_HTML.png)
![quantifications of western blots with imagej quantifications of western blots with imagej](http://www.lukemiller.org/journal/journal_pics/Westerns/Img11_box1.gif)
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… 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