![]() 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. This is what you get when you treat each row in the dot blot as a horizontal 'lane' and use the gel analysis procedure in the ImageJ manual. 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. This dot blot image is available in the File/Open Samples menu in ImageJ 1.33s or later. 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. In fact, the gel for the wild type was accidentally loaded with more of the sample. 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.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. The accuracy of this digital data is directly proportional to the bit depth of the imaging system. 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. This video describes how one can quantify their western blots through ImageJ. When a western blot image is captured, it must be digitized to convert the continuous tone intensity of the blot into digital data that encodes the intensity levels of each pixel. The report will automatically pop up on the side. A western blot image is made up of pixels, which contain information about how much signal was collected at each location in the. A protein band is a feature that appears in a western blot image. Furthermore, it can provide a qualitative assessment of changes in protein expression levels in response to stimuli or interventions. Note: western blots are semi-quantitative so they are ideal for showing relative protein expression rather than an absolute quantity. Ah yes, I mean to normalise my protein of interest against ponceau rather than a housekeeping protein such as gapdh. Image capture software and Image formats. You should do the western blot with your antibody and then quantify. The housekeeping protein taken here is -tubulin. It exhibits three lanes and four proteins as A, B, C and D. Traditional western blotting is useful for detecting the presence or absence of a specific protein in a lysate or complex mixture. The western blots are taken from Aquatic Ecology and Fish Biology Laboratory, Department of Zoology, Visva-Bharati University. 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. Key steps for achieving sensitive chemiluminescent quantitative western blots. Western blotting is routinely used to detect proteins and their posttranslational modifications (PTM) in biological samples. This protocol will allow you to relatively (no absolute values) quantify protein bands from western blot films. 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. Western Blot Image Analysis: Lane and Band Tools. 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. 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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