Develop a Western Blot Gel for a Protein of Interest
- 1). Thoroughly wash the PVDF membrane in TBST solution. Place the membrane in a rectangular container filled with TBST and place it on a tilt or rotation shaker at low speed (80 to 90 hertz) for 10 minutes. Dump the TBST out and repeat this wash.
- 2). Wash the membrane in blocking buffer. Carefully pour the TBST solution out and pour an equivalent amount of blocking buffer into the container. Place the container on a shaker at low speed for one hour.
- 3). Mix up your primary antibody while waiting for the blocking buffer to complete. Check your antibody to see what dilution is recommended by the manufacturer and dilute accordingly. For instance, if your antibody needs to be mixed in a 1:5,000 dilution, you can measure out 10 ml of blocking buffer and add 2 µl (microliters) of primary antibody.
- 4). Pour out the blocking buffer and add the primary antibody solution to the container. The amount of time needed to incubate the primary antibody with the PVDF membrane will vary with the antibody and tissue sample. The most common procedure is to place the membrane on a shaker at slow speed at 4 degrees C (in a refrigerator) for 16 to 18 hours. Your antibody should come with a recommended incubation time.
- 5). Rinse the PVDF membrane. Pour the primary antibody out and rinse the membrane for 10 minutes in TBST at room temperature. Repeat this washing step two or three times.
- 6). Incubate the membrane in secondary antibody. Your secondary antibody comes with a recommended dilution. For instance, if the recommended dilution is 1:10,000, you can add 1 µl to 10 ml of blocking buffer. Make sure your secondary antibody is labeled "HRP-conjugated" because other antibodies may not work properly with ECL kits.
- 7). Develop the PVDF membrane using ECL or a comparable chemiluminescence system. Check the directions that come with your ECL kit to see how to mix the developing solution and how to properly develop the membrane.
Develop the PVDF Membrane
- 1). Expose a film to the membrane. Within 10-15 minutes of ECL developing, bring the PVDF membrane to a darkroom. In complete darkness, expose a blank film to the membrane and place it in the darkroom's developing machine.
- 2). Once the film comes out of the developer, turn the lights on and see whether the film is adequately developed. If the film is too light, you will need to expose a new film for a longer time. If the film is too dark, you can try exposing another film for one minute or 30 seconds.
- 3). Note the exposure time that created a well-developed film. You can use this time for all subsequent runs of this procedure.
- 4). Scan the developed film into a computer using your scanner. With most scanners, you can place the film on the scanner bed and push a button. Consult the documentation that accompanied your scanner to determine if this is the proper procedure. Once the film is scanned in, save the image with a descriptive file name that you will be able to recognize later.
- 5). Open the saved image in the ImageJ program. ImageJ is developed by the National Institutes of Health (NIH) and is available for free online.
Compare Bands on the Developed Films
- 1). Obtain a background measurement. Make a rectangular selection around the largest band in the Western blot using the selection tool (the first icon in the toolbar). Then move the rectangle to an empty, undeveloped area of the film. Hold down Control+M to measure the background darkness.
- 2). Move the rectangle to the first band on the film and press Control-M again. Repeat this in sequence for all bands on the film that you wish to analyze.
- 3). Copy the band data. When you start measuring, a "Results" window will appear. Once you're done measuring, highlight all of the measurement data and press Control-C to copy it. Open up your spreadsheet program and press Control-V to paste the measurement data. Carefully label all pasted data and save the data file when you're done measuring all films.
- 4). Determine the density of each band. The value for your background measurement should be the highest number since it is brighter than the shaded bands of your Western blot film. Subtract the band measurement values from the background value to determine their relative density. For instance, if your background value is 200 and you have four bands with brightness values of 150, 170, 180 and 190, then the final values for those four bands are 50, 30, 20 and 10.
- 5). Compare experimental groups using whatever statistical analysis you wish to apply. Most experiments use a technique called ANOVA (analysis of variance) to compare values across groups. Many statistics packages such as PSAW have built-in statistics procedures to run ANOVAs on properly formatted data.