imagej western blot quantification protocol

#Imagej western blot quantification protocol software

Select the ladder image and set the opacity to ~75%. To position the ladder, return to FIJI and click on the blot image, then overlay the ladder (Image > Overlay > Add Image).Using Screen Capture (shift-ctrl-command-4), take a snapshot of both the ladder image and the blot image and paste them into powerpoint.Clear everything outside of the ladder in the ladder image by drawing a rectangle around the ladder lane then use Edit > Clear Outside.Be sure to rotate both images exactly the same Use Grid Lines = 20, Bilinear interpolation and click the preview box.

#Imagej western blot quantification protocol software

Software algorithms determine the density of signal across a selected area. Detection methods include colorimetry and immunoblotting (via chemiluminescence, fluorescence, and radiolabeling). Rotate the images so the bands are in a horizontal line (Image > Transform > Rotate) then adjust Angle until bands look horizontal. In Western blotting, densitometry quantitates proteins within the linear dynamic range of a chosen detection method.Adjust both the blot image and the ladder image (Image > Adjust > Brightness/Contrast) so that they look good to you.Generally invert the blot image so that bands are black and background is white (Edit > Invert).Open the blot image and the ladder image and convert both images to 8 bit (Image > Type > 8-bit).Transferring images to powerpoint using FIJI (ImageJ) Figure conclusion: Short paragraph with your conclusions.Figure caption: Short paragraph describing Western Blot method.(change words in red words to your specific experiment) Label Y-axis: Protein expression relative to control lysate normalized to housekeeping gene.Label band(s) of interest with protein name.Label every lane (optional: use numbers and have a number key below).Filenames of the original images on proteinsimple machineīlot image used for densitometry measurements.Thumbnails of the entire images of the blots.Subtitle: Your initials, and date where details can be found in your lab book (see Lab Book Details). Title: Date, protein(s) and cell lysates including conditions being analyzed. 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.When presenting a western blot in a Starr lab meeting or presentation, include the following information: 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. In fact, the gel for the wild type was accidentally loaded with more of the sample. 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.