Quantitative Western Blotting to Estimate Protein Expression Levels

For electrophoresis, set up a pre-cast 4 to 12% Bis-Tris gradient gel in the gel electrophoresis chamber system, and load 3.5 microliters of a protein standard into the well. When using an internal standard for between membrane normalization, load an amount that is equal to the other samples into the first three wells next to the protein ladder, and load 30 micrograms of each sample into the remaining wells. Then, run the samples through the stacking gel at 80 volts for 10 minutes followed by 150 volts for an additional 45 to 60 minutes.

At the end of the electrophoresis, to assemble the transfer stack, place the protein gel onto the bottom stack containing the polyvinylidene difluoride membrane followed by the filter paper. Use the blotting roller to remove any air bubbles, and place the top stack on the top of the filter paper, before rolling the stack again to remove air bubbles.

Transfer the whole stack into the transfer device with the electrode on the left side of the device and place the gel sponge on top of the stack, so that the sponge is aligned with the corresponding electrical contacts on the device.

After closing the lid, select and start the appropriate program. At the end of the program, cut the membrane to the gel size, and wash the cut membrane quickly with double-distilled water before continuing with the total protein stain.

For total protein staining, roll the membrane into a 50-milliliter tube with the protein-side facing inwards and label the membrane with 5 milliliters of protein stain solution on a roller for 5 minutes at room temperature in a fume hood.

At the end of the incubation, wash the membrane quickly with 5 milliliters of wash solution, returning the tube briefly to the roller between washes, followed by a brief rinse with ultra-pure water. Add 3 millimeters of blocking buffer to the membrane, and return the membrane to the roller for 30 minutes at room temperature. Replace the blocking buffer with the primary antibody of interest at the appropriate optimized concentration, and incubate the membrane on the roller overnight at 4 degrees Celsius.

The next day, wash the membrane 6 times for 5 minutes with 5 milliliters of fresh PBS per wash on the roller at room temperature. After the last wash, incubate the membrane with the appropriate secondary antibody solution on the roller for 1 hour at room temperature followed by 3 washes for 30 minutes per wash. After the last wash, dry the membrane, and use aluminum foil to keep the membrane protected from light.

For image acquisition, place the membrane on the scanner with the protein-side facing down and select the scanning area in the software. Then acquire images in both channels and export the images to an appropriate image analysis program.

Display the 700-nanometer channel to show the total protein staining result, and select "Analysis" and draw a rectangle to define the area of interest for normalization. Then, copy and paste the first rectangle area onto each individual sample to ensure the defined region is the same size for all of the analyzed lanes.

To quantify the protein concentration in each lane, copy the results from both the total protein stain and the protein of interest to a spreadsheet program, and determine the highest total protein stain signal. Then divide each total protein stain signal value by this value to obtain the normalized protein loading value, and divide the 800-nanometer signal value from each individual sample by its corresponding normalized protein value to calculate the relative protein expression ratio in different samples.