The technique of western blotting involves several key steps. Briefly, it involves sample preparation, gel electrophoresis of the isolated proteins from the samples, followed by transfer of the proteins onto a more durable membrane (nitrocellulose or PVDF). The membrane is then blocked to avoid nonspecific interactions, in the subsequent step of adding the target protein antibody.
Following incubation of the membrane-bound protein with the target antibody, the presence and quantity of the protein (s) is verified.
For a detailed walkthrough of how to quantify the detected protein on the nitrocellulose or PVDF membrane, see my YouTube video embedded below.
Quantifying western blot bands using image analysis software, is an essential step to determine the relative expression levels of proteins. There are several software that may be used. Common options include Image J (Fiji), GelAnalyzer and proprietary software from imaging systems such as BioRad's Image Lab. Below are the expanded steps to perform accurate quantification in ImageJ.
Step 1
Obtain a high-quality image: use a digital imaging system or scanner to capture the blot. Ensure the image is well-exposed and not over-saturated.
Step 1.1
Format and Resolution: save the image in a high-resolution format (e.g., TIFF) for better accuracy during
Step 1.2
Prepare the Image for Analysis:
Transform the gel image if you find that it's upside down or it's not in the right orientation after opening the file. To do this, go to Transform and rotate to flip it right around.
Step 1.3
Convert the image to Grayscale: convert the image to grayscale, as most analysis tools work on intensity values in black-and-white images.
Step 1.4
Adjust the Brightness and Contrast: optimize brightness and contrast for clear visualization of bands without introducing artifacts.
Step 1.5
Crop the Image: crop unnecessary parts of the image to focus on the lanes and bands being analyzed, avoiding marker lanes if not relevant.
Step 1.6
There is an option to invert the image. This allows the peaks that you are about to detect and quantify, to appear upside down, so that you can draw a line at the base of the peak for quantitation.
Define the Region of Interest (ROI)
Step 2
Mark Bands: Draw rectangular or elliptical ROIs around each band you want to quantify. In ImageJ, I use the same rectangle to quantify each of the bands. To enable this, I first move the rectangle to all the bands and adjust it to ensure that it's the right width and height to capture each band.
Step 2.1
Measure band intensity
In ImageJ, you do so by selecting --> Analyze. In Analyze you go right down to the specific option which says ---> Gels and then you can select the first lane.
After selecting the first lane, use the forward arrow on your keypad and it will duplicate that rectangle.
Move to the next band/lane with the duplicated rectangle, then select ---> Analyze ---> Gels.
This time, you must choose --> 'select next lane' rather than 'select first lane'.
Repeat with --> 'select next lane' while using the arrows on your keyboard to move to all the bands that you want to quantify.
Once you've quantified all the bands using the duplicated rectangular tool, you want to Plot Lanes.
Step 2.2
Go back to --> Analyze --> Gels and then this time, choose ---> Plot Lanes. The software will now plot the peaks to match the intensities it has detected in each lane.
Once you have your peaks you want to draw the boundary of each peak. This is why it is useful to have inverted the image. With the inverted image, the peak will appear upside down, allowing you to see the boundaries of the peak clearly.
Draw a line to demarcate where a peak begins and ends.
Once you have the peak clearly marked, you select the wand tool.
Using the wand tool, click on the peaks that you have drawn, to obtain the pixel intensity within each band.
Correct for Background: measure an empty region near any of the bands to use for background correction.
Copy the numerical output of each peak (and the background peak) and paste the outputs in Excel or your data analysis tool of choice.
Subtract the background intensity value from each of the peak intensities.
Normalise data using the loading control: normalise band intensities to a housekeeping protein such as beta-actin or GAPDH to correct for variations in loading and transfer.
Relative Quantification: divide the band intensity of the target protein by the intensity of the loading control in the same lane.
Plot and Analyze Results
Statistical Analysis: perform statistical tests such as t-tests or ANOVA to determine the significance of differences between groups.
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