Analysis on Molecular Gels: A Case Study in Polyacrylamide Gel Electrophoresis (Procedure) : Biological Image Analysis Virtual Lab : Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab

	Theory Procedure Self Evaluation Simulator Assignment Reference Feedback

For informations about installation of the image analysis tool, access the “Simulator” tab

Step 1: Open ImageJ by double clicking ImageJ icon from the desktop or one may go to start programme ImageJ on the windows operating system.

ImageJ will open in a new window as shown below.

Step 2: To import an Image into ImageJ, Goto files ---> Open. A popup menu will appear. Use Choose button to choose your particular images into ImageJ software. The imported picture will shown in a new window.

Step 3: Adjust the color of image to "grayscale” (go to image ---> Type ---> 8-bit).

Step 4: For inverting image for convenient viewing and economic printing, Go to Image ---> Lookup Tables ---> Invert LUT. One can use Brightness and Contrast enhancement once again, but this one is good enough to move on.

Step 5: Choose the Rectangular Selections tool from the ImageJ toolbar. Draw a rectangle around the first lane. Use mouse to click and hold in the middle of the rectangle on the 1st lane and drag it over to the next lane or can use the arrow keys to move the rectangle. ImageJ will automatically align the rectangle on the same vertical axis as the 1st rectangle in the next step. Press 2 (Command + 2 on Mac) or go to Analyze ---> Gels --->Select Next Lane to set the rectangle in place over the 2nd lane. The number 2 will appear in the lane when the rectangle is placed.

Step 6: Select Analyze ---> Gels --->Plot lanes. Plot gives the relative location of each band and banding intensity (a measure of enzyme activity)

Step 7: Click on Analyse --->Measure to quantify the selected area. A new window that appears which will helps to find the measurements for Integrated Density, Area and Mean Gray Value, etc. Repeat the above steps until all bands have been measured. Then copy all the values to a spreadsheet. The quantified images are now ready for data analysis in the spreadsheet program.

Result Interpretation:

The plot obtained from the imageJ gives the relative location of each band and banding intensity. The “results” window contains the area and mean which indicates the intensity of the band as a numerical value – the brighter the band, the higher the number.

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