![]() I have very little experience using Scion Image but, when using the Gelplot Macro, I can only select the different lanes with the rectangular selection tool and the size of the rectangular selection has to stay the same for each lane. With results like the ones illustrated on the image, wouldn’t it be better to take into account the size (area) of the band and not only its intensity ? Here is an illustration of the results I get: Also if anyone can improve my described method, dont hesitate to do soĪnother one on western blot quantification and Scion Image (or ImageJ)… Next subtract the background value (area x mean intensity) from each of your band values (area x mean intensity) I might be wrong, but in order to quantify your bands, youll need to multiply the mean intensity and the area together to get the number you want. Use care in making your ROIs and I'd experiment drawing ROIs and comparing the values you get from them until you think you can draw them properly and get an accurate measurement. Repeat this for all the bands you want to measure. Again youll get an area measurement, and a mean intensity measurement. Next, draw a ROI (region of interest) around a band that you want to measure and select analyze-> measure. The technique was developed in 1979 1 by Harry Towbin and colleagues and later named the western blot due to the technique’s similarity to Southern blotting. This will tell you the background intensity of your image (mean), and also the area of your box Write these numbers down. A western blot, sometimes called a protein immunoblot, is an antibody-based technique used to detect the presence, size and abundance of specific proteins within a sample. Im sure this method is far from perfect, but It might work for youįirst draw a box in an area that has no bands and select analyze->measure. Ive never used scion image, but ithe pprinciple i suppose would be the same in any image program. I dont have any experience using photoshop to quantify the bands as well. We scan our films in grayscale and produce a 1200 dpi resolution TIFF file.ĭo not make a jpeg of your film, jpegs contain far less image data then tiff files. Just remember when you make a digital image of your film using a scanner, you need to create the highest quality image possible. This is a VERY rough way of doing it, but do give you some hints on the relative increase/decrease of expression. ![]() This way to don't have to take the box size into consideration)Ĥ) Then do, Image>Histogram and write down the Meanĥ) Then drag the box to the next band and repeatĦ) Finally, drag the box to measure the Mean from the Background, and subtract the number from all the others.ħ) Divide all the numbers with your reference number for the one set as 1 (or 100%) The quantification is all relative towards one band you put as 1 (or 100%).Ģ) Then do, Image>Adjust>Invert (this way, you invert the scale)ģ) Draw a box, which covers ONE band using the Marquee Tool (do the biggest band first, as the box HAS to be the same size all way through the analysis. Secondly, ALL the bands you want to compare have to on the same film. put it up against a piece of paper and see if you can read soem text through it.) Rule of thumb is you have to be able to see through the bands (i.e. ![]() First thing you have to do is get a GOOD exposure of your gel. ![]()
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