#Western blot quantification with imagej software

Save your Results window so that you can transfer the measurements to excel to generate a standard curve. A window will pop up with a threshold and the color options allowing for the selection of threshold values based on the nature of the image and fluorescence intensity. On the ImageJ interface, select the 'magic wand' button and then click on the line defining the area of the curve of the first standard, and the areas of the curves in your protein analysis lanes. Click on the ‘Image’- go to the ‘Adjust’ tab-click on the ‘Threshold’ button. If you are not working within the linear range, e.g., if your detector or film. Perform steps 1 and 2 as described in Approach 1 (3.2.1).

#Western blot quantification with imagej software

In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The Steps for Western Blot Quantification (Quantitative Western Blot protocol) Find the Linear Range For quantitative analysis of an image you must ensure your image was captured in a manner sensitive enough to detect change, in what we call the linear range. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. However, this method could be highly subjective and presents a variation source when non-uniform background is obtained. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. The usual method for Western blot quantification by ImageJ involves determination of a rolling ball radius value that adjusts the best background subtraction under the observer criteria. Use the square selection tool to highlight the first. 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 (ImageType8-bit). It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Since its first description, Western blot has been widely used in molecular labs.