![]() ![]() 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. 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. Image can handle scans somewhat larger than these, but you will probably run into problems if you greatly exceed these recommended sizes. This study presents a protocol of image analysis. Figure shows a stylised western blot of increasing concentrations of protein, and the signal intensity as measured by a. In literature, few studies have been reported using image analysis for the quantification of protein in SDSPAGE: that is, imaged with StainFree technology. 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 measurement of the areas will be bumped to a 'Results' window. Continue selecting the area outlines of the remaining lanes. The report will automatically pop up on the side. The protein purity is generally checked using SDSPAGE, where densitometry could be used to quantify the protein bands. 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. 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. 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. 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. 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. 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. This dot blot image is available in the File/Open Samples menu in ImageJ 1.33s or later. Use the square selection tool to highlight the first lane. Electrophoretic gels such as Western blots need frequently to be quantified in order to translate biochemical results into statistical values (see Gels ). Collectively, the results of these two studies suggest that specific proteins may be evaluated by using relatively inexpensive image analysis software systems via pixel quantification of electronic images.Convert the image to 8-bit using ImageJ function (Image→Type→8-bit). Same tools can be used for DNA gel images, thin layer chromatography, Southern blot. A second image analysis program, Alpha Imager 2000, was then used to define the boundaries of protein bands, assess pixel number and density, and to obtain final numerical data for quantifying alpha-Actinin on the WB. This demonstrates how to create a figure to display western blot results. In the second procedure, WB were scanned with a ScanJet 3c flat bed scanner and their backgrounds were clarified using Image-Pro Plus. Dot blots corresponding to a linear concentration range from 10 to 300 ng IGF-I were assessed by this method. After the DB were developed and dried, the images were digitized using an HP Deskscan II flat bed scanner, exported into Image-Pro Plus and analyzed by taking the combined mean of 45 degrees and 135 degrees sample lines drawn through each dot. 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. In the first procedure, known IGF-I samples were dotted on nitrocellulose membranes using a vacuum manifold. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and alpha-Actinin on Western blots (WB). ![]()
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