Quantitative Western Blotting to Estimate Protein Expression Levels

Quantitative western blotting — a total protein normalization-based technique — is useful for quantifying target protein expression levels.

To begin, load tissue lysate into the wells of a polyacrylamide gradient gel. The lysate contains the target protein, along with other cellular proteins.

Run the gel at an appropriate voltage for the electrophoretic separation of the sample proteins along the gradient. Upon completion, place the gel on top of the polymeric membrane of a transfer stack.

Apply an electrical current to transfer the separated proteins onto the membrane. Next, place the membrane in a fluorescent total protein stain. The charged dye molecules in the stain bind electrostatically to all the proteins on the membrane.

Add a blocking solution. The proteins in the solution bind to the membrane, thus. preventing the non-specific binding of detection reagents. Add a primary antibody binding only to the target protein. Then, add a secondary antibody — labeled with a fluorophore — that attaches to the primary antibody.

Excite the sample with a laser. Record the emitted fluorescence from the dye molecules of the total protein stain and the antibody-bound fluorophores at their corresponding wavelengths.

To calculate the normalized protein loading value, divide the total protein signal of each lane by the highest value. Divide the target protein signal by the corresponding normalized loading value to determine the relative expression of the target protein.

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.