![]() ![]() P.S: I incubated these membranes with primary antibody A, read it, then primary antibody B, read it, then lastly with housekeeping gene. Analyzing Electrophoretic Gels Use the Calibrate command to calibrate the image to a calibrated optical density step tablet. Thus, I am now very confused of which program is more reliable. And my PI does not agree that ImageJ makes more sense than Odyssey. HOW TO QUANTIFY WESTERN BLOT BANDS using ImageJ Area Under The Peak Method Adwoa Biotech. This time the numbers are much similar to each other, but I still don't understand how the area and percent work and what they really mean. Then I tried using ImageJ to re-do all measurements. Western blotting was first introduced in 1979 as a means of detecting a target protein in a sample with antibodies and detection using film 1,2. If even the housekeeping gene readings are so different, how can we expect the soluble protein readings to be accurate? (this is a question) For example, I ran 40ug of proteins for all samples, but I would get an average intensity reading of 80 on one membrane and 120 on another for the housekeeping gene. Quantification of western blot using imageJ for beginners western blot quantification imagej Biology Lectures 22.9K subscribers Subscribe Subscribed 805 Share 72K views 2. However, no matter how similar the procedures were, there was still some huge difference in band expression. This protocol offers an Image J macro/plugin that enable easy quantification of bands on western blots, dot blots, and fluorescent gels etc., by simply. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. We are trying to compare between 2 sets of 8 samples, so I ran 2 western blots simultaneously all the time. Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. Not sure if many people are familiar with the Odyssey software, but we use it to scan our membrane and to quantify the bands. If you want a more sensitive method than ponceau you can try 2,2,2-Trichloroethanol ( https. I am currently using IR-dye secondary antibodies purchased from LI-COR to detect some soluble proteins. Use the entire lane instead of one band, otherwhise you are still normalising based on just one protein. Hi all, this is my very first project and I hadn't have any experience at all. ![]()
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