using imagej for western blot quantification

Using imagej for western blot quantification how to#
Using imagej for western blot quantification full#

Using imagej for western blot quantification full#

Recent articles have dissected and contrasted the old practices passed down from the early days of western blotting to offer revised methodologies that take full advantage of the currently available state-of-the-art instruments and reagents to achieve accurate, precise and reproducible data 14, 15, 16. The associated retractions from high profile articles 10, 11, 12, 13 support the unfounded reputation of western blotting as a difficult, frustrating and non-reproducible technique that is semi-quantitative at best. However, the original qualitative techniques and assumptions that have been passed down through generations of lab members were not formally updated to permit the production of semi-quantitative western blot data thus giving poor reproducibility and misleading interpretations 7, 8, 9. This permitted the generation of semi-quantitative data from chemiluminescence. During this time, the first camera-based imaging systems were introduced which enabled automated detection over a much broader linear dynamic range of about three to four orders of magnitude 6. Since the linear quantitative range of film is only about an order of magnitude and very challenging to assess saturation by eye, the results even between technical replicates were difficult to reproduce 5. Over the following two decades, as reagent and antibody technologies evolved, scientists attempted to produce quantitative results initially by scintillation counting and film densitometry 3, 4. Western blotting was first introduced in 1979 as a means of detecting a target protein in a sample with antibodies and detection using film 1, 2.

Using imagej for western blot quantification how to#

The data reveal when and how to apply these detection methods to achieve reproducible data with a stepwise approach to data processing for quantitative analysis. A methodology to optimize and produce excellent, quantitative western blot results with rigorous data analysis from membranes probed with both fluorescent and chemiluminescent antibodies is described. Fluorescence reagent and detection solutions are the latest innovation in western blotting but there remains questions and debate concerning their relative sensitivity and dynamic range versus chemiluminescence. The development of sensitive, camera-based detection technologies coupled with an updated technical approach permits the production of reproducible, quantitative data. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate and reproducible quantification has been a major challenge from this methodology. This will produce an image that will allow you to align position your ladder image next to your blot image in powerpoint.Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. 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. Rotate the images so the bands are in a horizontal line (Image > Transform > Rotate) then adjust Angle until bands look horizontal.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. When presenting a western blot in a Starr lab meeting or presentation, include the following information: