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Quantification of Protein Present in a Sample
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For informations about installation of the image analysis tool, refer “Simulator” tab

 

Step1: Import the  western blot image into the image analysis software.

 

 

The image will open in a new pop up window as shown.

 

 

Step 2: Choose the rectangular Selection tool from the toolbar.

 

 

Step 3: Draw a rectangle around the first lane of the western blot image.

 

 

Step 4: When the first band is selected in a rectangle, then select Analyze ---> Gels ---> Select first Lane from the menu bar (or “control-1″ in Windows)

 

 

The image will be displayed as shown below.

 

 

Step 5: After selecting the first lane, take the cursor and move it within the box set in the first lane. This helps to click-and-hold, and drag the box to the second lane.

 

 

Step 6: Then analyze the gel. Select Analyze---> Gels---> Select Next Lane or  (“control-2″ in Windows). This will select an area in the second lane that is exactly equal to the area selected in the first lane.

 

 

The image will be displayed as shown below

 

Step 7: Repeat the steps 5 and 6 to select the band in the third lane. Click within the yellow box and drag the box to cover the next band.

 

 

Step 8: Then to analyze, follow, Analyze---> Gels---> Select Next Lane or the click “control-2″ in Windows.

 

 

The image will be displayed as shown below.

 

 

Step 9: After selecting the last lane, select the Analyze-->Gels---> Plot Lanes menu in the menu bar  or the click “control-2″ in Windows.

 

 

A new pop window will open which depicts the densitometry measurements of the western blot gels. This graphically depicts the intensity of the selected bands in the gel.

 

 

Step 10: Using the line tool close off all of the peak area that rises above the background level.

 

 

 

Step 11: Select the wand tool and click inside each peak, starting from the top. The peak will change into yellow color.

 

 

 

This depicts the area under the curve for each peak.

 

Step 12: Another window that depicts the “Results” will open that will help to analyze the band intensity.

 

 

These values can then be used to make relative comparisons of band intensity between lanes. This comparison is an indication of the relative amounts of protein that were loaded into each well.

 

Step 13: Copy and paste the values in an Excel Spread sheet and find out the relative values.

 

To calculate the relative values, simply divide each value of the bands with a standard value. 

 

Here, 9069.066 are taken as the standard value.

 

7209.409/9069.066   =   0.7949

9069.066/9069.066  =   1

27932.057/9069.066  =  3.08

19621.874/9069.066 = 2.16 

 

Result Interpretation:

 

Compare the relative quantities of proteins present in the sample. Here, the  result indicates that the band in the third lane is more intense compared to other bands in the gel. This means that the amount of protein is more in the third lane compared to other lanes in the analyzed gel image.

 

 

 

 

Cite this Simulator:

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