Running a duplicate protein gel and developing with Coomassie stain 5 can help to remove this uncertainty as it will show the amount of total protein 6 in each sample lane and can reveal any loading inconsistencies. This could mean that there is twice as much of the target protein in that sample, or it could mean that more sample or a more concentrated sample has been loaded in one lane than the other. For example, when assessing a blot, the band from one sample may appear twice as bright as another sample. It is essential, especially when trying to compare protein expression between different samples, to know how much sample has been loaded as this may not be apparent from the blot alone. It is also important to load appropriate control samples and size marker ladders to enable interpretation of the final blot.
Solids will impair the running of the gel and it is likely your protein of interest will remain in the stacking gel. If your protein of interest is in the insoluble fraction (e.g., cell membrane-bound proteins) investigate pretreatment methods to liberate and solubilize it first. For a clean image, samples are centrifuged to remove any solids, in order to load only the soluble fraction. The specific separation method chosen will depend on the aim of the analysis. This is typically achieved by protein electrophoresis, such as sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) or native PAGE, which separates proteins based on their molecular weight or charge. Credit: Technology Networks Western blot gelīefore a western blot can be performed, the proteins in the sample must be separated. 3, 4įigure 1: Overview of a western blot protocol. The second contains the results obtained by doing background correction using Process/Subtract Background, selecting" Integrated Density" in Analyze/Set Measurements, and then measuring each of the 28 dots using a circular selectionįigure 4. Circular selection and integrated density measurements Figure 5.Figure 1 shows an overview of a western blot protocol. The first column in this spreadsheet contains the integrated densities (as a percentage of the total) of the 28 dots measured using the gel analysis procedure The "Use Inverting Lookup Table" option in Edit/Options/Image will invert the pixel data and invert the lookup tableĥ. You can invert the lookup table (Image/Lookup Tables/Invert LUT) to restore the original appearance of the image. Notice how the image now has a black background? It was inverted (Edit/Invert) so background pixel values are near zero, which is required for correct calculation of integrated density. After correcting the background, enable "Integrated Density" in Analyze/Set Measurements, create a circular selection, drag it over the first dot, press "m" (Analyze/Measure), then repeat for the other 27 dots. This method usually requires background correction of the image, which can be done using the Process/Subtract Background commandĤ. In the second method, you measure the integrated density of each dot by outlining it and using the Analyze/Measure command. Profile plots Figure 3. Profile plots of first rowģ.
"Invert Peaks" in the Analyze/Gels/Gel Analyzer Options dialog was enabled to avoid having upside down peaksįigure 2. 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 manualĢ. The numbers on each peak are the size of the corresponding dot as a percentage of the total size of all the dots. The first is to treat each row as a horizontal "lane" and use ImageJ's gel analysis functionĢ. The second is to subtract the background and measure the integrated density of each dotġ. There are two built in methods for analyzing a dot blot in ImageJ.ġ.