This protocol can be used to address important questions in fundamental and clinical research by looking at protein expression across different tissues and time points. To perform reliable quantitative Western blotting, we combine a fluorescent total protein stain with an internal loading control. This overcomes limitations that arise when comparing various tissues across experimental conditions.
To extract proteins from snap-frozen cell or tissue samples, thaw the minus-80-degree samples on ice before washing the samples as appropriate, according to the table. Using a handheld electric homogenizer with a polypropylene pestle, homogenize the washed samples, rinsing the pestle in double-distilled water and drying with a clean tissue between samples. Leave the samples on ice for 10 minutes, followed by centrifugation.
Then, transfer the protein-sample-containing supernatant to a new tube on ice without disturbing the pellet. For quantification of the protein concentration, set up bovine serum albumin standards at increasing concentrations in triplicate, and add one microliter of each protein sample in duplicate to the appropriate wells of a 96-well optical plate. Incubate the protein on a 60-degree-Celsius heat block for 10 minutes or longer if the protein concentration is expected to be low and measure the absorption at 560 nanometers on a plate reader.
Export the plate reader measurements and calculate the protein concentration by comparing the average absorbance values of each sample to a standard curve obtained using the protein standard. To normalize the amount of protein, prepare dilutions of the protein samples in sample buffer and ultra-pure water and incubate the samples in a 70-degree-Celsius heat block for 10 minutes. Then, place the samples on ice before vortexing and briefly centrifuging.
For electrophoresis, set up a precast four 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 five milliliters of protein stain solution on a roller for five minutes at room temperature in a fume hood.
At the end of the incubation, wash the membrane quickly with five milliliters of wash solution, returning the tube briefly to the roller between washes, followed by a brief rinse with ultra-pure water. Add three milliliters 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 four degrees Celsius.
The next day, wash the membrane six times for five minutes with five 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 one hour at room temperature followed by three 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 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.
In these representative Western blots, proteins extracted from tissues obtained from post-natal day five mice are compared to proteins extracted from 10-week-old adult mice. Quantification of the fluorescence intensity of the total protein stain was achieved by measuring the fluorescence intensity inside the rectangle box on each lane. When whole lanes are analyzed, the fluorescence intensity remains relatively similar across samples, indicating that using the total protein stain for normalization is suitable for this purpose.
The fluorescent total protein stain can also be used to compare protein levels at different developmental time points. For example, although survival motor neuron protein levels clearly decrease with age in mice, the total protein stained quantification remains constant. The use of internal standards, fluorescence-based normalization, and adequate statistics increases the robustness of complex protein expression analysis across different conditions.
This protocol adds to traditional Western blot techniques, overcoming the issues of appropriate loading controls and comparisons across experimental batches, and providing flexibilities for protein expression analysis. This approach can be extended to comparing protein expression under other experimental conditions.
