P.S: I am aware it's better to normalize to total protein. I don't know the reason why this happens, and i can't explain it to google so i couldn't find my answer :(Īttached are some images in case no one understood my gibberish. When i do step 3, imagej changes the grey value of the copied image (the image either appears lighter or darker when i do that). Purpose: This protocol describes how to quantify any type of cellular foci, such as phosphorylated Ser139 on histone variant H2A. That’s why I posted it here Everything I have found in the web so far was mostly going a in the wrong direction leading to non. 1: Many students try to find a guideline for Western blot quantification online while using ImageJ. I also tried the following in order to encounter the problem (not sure if it's right.)ģ-Select the bands, copy them and paste them to the GAPDH image And I think the topic is important in the aspect of reliability and reproducibility in science. SO, how do I normalize to GAPDH in this case? Is it really okay to compare images produced by different exposure times? Use two different software tor quantification. Optimize quantification using well established antibodies and sample lysates. Some proteins say, GAPDH, produces a good signal in less than 1 second of exposure. 7,19 Quantification Densitometry analysis to target protein using afferent software : Inaccurate quantification to protein to interest, poor image quality, and signal saturation. So after adding the ECL ( enhanced chemiluminescence )substrate to my membranes, I detect using the imageQuant system, usually, I expose the membrane for different time periods until I see a good signal. Three scanning modes are supported: 8-bit grayscale, 8-bit indexed color and 24-bit color. I am new to using ImageJ (Fiji) and I am trying to relatively quantify the density of my bands (using mean grey values) Using Image with Flatbed Scanners The Acquire command provides direct support for most scanners that have Photoshop plug-ins. Go to Analyze>Gels>Gel Analyzer Options and click the boxes for Label With Percentages, Outline Lanes and Invert Peaks.
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