The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability.īackground subtraction Densitometry ImageJ Quantification Western blot.Ĭopyright © 2018 Elsevier B.V. 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. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. An image of the membrane is then capturedeither on to film or using a digital imagerto allow for analysis. Western blotting begins with separating a mixture of proteins on a gel, transferring them to a membrane, and detecting one or more targets of interest with labeled antibodies. 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. Basic principles of western blot image analysis. Ah yes, I mean to normalise my protein of interest against ponceau rather than a housekeeping protein such as gapdh. One broadly used program to quantify images of western blot bands is the Scion Image Software (Scion, Frederick, MD) Cite. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. You should do the western blot with your antibody and then quantify. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Since then, it has been mentioned in the titles, abstracts, and keywords of more than 400,000 PubMed-listed publications. Figure 2 shows a stylised western blot of increasing concentrations of protein, and the “signal intensity” as measured by a commonly used software-in this example the last five concentrations gave the same intensity measurement despite representing very different amounts of protein.Since its first description, Western blot has been widely used in molecular labs. The Western blot technique was first published 40 years ago. This represents a general problem of quantifying western blots with simple image analysis software, which may be unable to discriminate between similar-looking bands that have fallen off the end of the linear scale. The chemiluminescent film was saturated, so the higher level of tubulin in the wild type was not reflected when the intensity measurements were taken: actually when the same amounts of sample were loaded, there was no change in expression of Protein X in the two conditions. The peak area is the signal used in most densitometry analysis (Gassmann et al., 2009 Gorr & Vogel, 2015 Rehbein & Schwalbe, 2015), and the peak maximum intensity has been used in the quantification of proteins in Western blots imaged by fluorescence (Grtler et al., 2013 Holzmller & Kulozik, 2016). Also, you cannot compare the results between blots, so if you have many samples, you need to run a standardized sample, or ideally several dilutions of a standardized sample, on each blot. then band intensity is in the linear range for the method used. 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. This protocol offers an Image J macro/plugin that enable easy quantification of bands on western blots, dot blots, and fluorescent gels etc., by simply selecting bands with rectangle or. Things to watch for: you should use blots that are not saturated, i.e. In fact, the gel for the wild type was accidentally loaded with more of the sample. Quantification of foci using Fiji or ImageJ. Blot Imaging and Densitometric Analysis The chemiluminescent blots were imaged rst with the ChemiDoc MP imager (Bio-Rad) and then on lm. However, although the two tubulin controls look the same-and give the same intensity measurements using a simple image analysis tool-they do not represent the same underlying expression. This article illustrates some of the easy ways to arrive at a false conclusion when trying to quantify protein levels from western blots.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |