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. 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. The report will automatically pop up on the side. Western blotting is a universally used technique to identify specific proteins from a heterogeneous and complex mixture. 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. Draw the line at where the peak begins and ends (bend in the line) for each peak. Use the line tool to draw the lines to eliminate the lane background from the calculations. 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. 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. After transforming a source image into an 8-bit greyscale image and inverting it in ImageJ/Fiji, the rectangle tool and ROI manager were used to define a series of regions of interest (ROIs), covering each dot with a single ROI of the same size, and the integrated density of each ROI was. Purpose: This protocol describes how to quantify any type of cellular foci, such as phosphorylated Ser139 on histone variant H2A.X (H2AX) stained images of tissue or cells, using the free NIH Image Processing and Analysis in Java software called Fiji or ImageJ. the blots were tracked and ImageJ version 1.52 software was utilized to. Use the square selection tool to highlight the first lane. Protein Quantification with Fiji and Microsoft Excel. Quantification of foci using Fiji or ImageJ. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Convert the image to 8-bit using ImageJ function (Image→Type→8-bit). In biological sciences, western blotting technique is widely used to quantify the expression of proteins in a given sample. First of all, you need to make sure you have done some kind of BCA assay to quantify the amount of total protein in each sample.
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