Since the area intensity is in arbitrary unit, it can also be normalised to the BCA assay measurement, DNA content or any other number chosen. Your gel will look something like this (see below). Once the gel image is open, you can zoom in with 'Ctrl+' and zoom out with 'Ctrl-'. You can drag the image you want to open onto the ImageJ window. To normalise the intensity of the area underneath the peak to the Ponceau staining, measure the intensity of 3 randomly chosen peaks on the Ponceau image, average the measurements and use that value to normalise the data against. Open ImageJ using the shortcut on the desktop. The report will automatically pop up on the side. Go to: Analyse→Gels→Label Peaks to get the report.Īlternatively, use the magic wand tool to highlight the area underneath the peak for each lane. Make sure that the image is in 8-bit mode: go to Image>Type>8-bit. In literature, few studies have been reported using image analysis for the quantification of protein in SDS-PAGE: that is, imaged with Stain-Free technology. We’ll use ImageJ’s gel analysis routine to quantify the density and size of the blots, and use the results from our loading-controls (lower bands) to scale the values for our protein of interest (upper bands). Draw the line at where the peak begins and ends (bend in the line) for each peak. The protein purity is generally checked using SDS-PAGE, where densitometry could be used to quantify the protein bands. Click the centre of the square and drag it across to the next lane. Press Ctrl and 1 to set first lane (Command and 1 on the Mac). Use the square selection tool to highlight the first lane. Use the line tool to draw the lines to eliminate the lane background from the calculations. Convert the image to 8-bit using ImageJ function (ImageType8-bit).
Continue this for the subsequent lanes (pressing Crtl and 2 every time).įor the last lane, repeat the procedure but press Ctrl and 3 to set the last lane. This protocol will allow you to relatively (no absolute values) quantify protein bands. Fundamentals of densitometry It is possible to use a scaling system for pixels, which has a one to one correspondence to the concentration of what you are studying. Press Ctrl and 1 to set first lane (Command and 1 on the Mac).Ĭlick the centre of the square and drag it across to the next lane. Quantifications of Western Blots with ImageJ by Hossein Davarinejad. This section will serve as an introduction to the program ImageJ and its use in the densitometric analysis of 1-D electrophoretic gels. Use the square selection tool to highlight the first lane. Use the square selection tool to highlight the first lane. Convert the image to 8-bit using ImageJ function (Image→Type→8-bit).
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