Quantitative Western Blotting to Estimate Protein Expression Levels

NOTE: This protocol has been optimized using standardized, commercially available kits and reagents in order to increase reproducibility (see Table of Materials).

1. Preparation of samples

1. Protein extraction
   1. Transfer snap-frozen cell or tissue samples from -80 °C on dry ice, thaw on ice, and wash as required with ice-cold 1x phosphate-buffered saline (PBS). Avoid unnecessary freeze-thaw cycles as this will affect protein quality.
   2. Add radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing 1x protease inhibitor to each sample, using the optimal amount per tissue weight.
      NOTE: Depending on the application, the type and amount of homogenization buffer may need further optimization.
   3. Use a hand-held electric homogenizer with a polypropylene pestle to homogenize tissue samples. Between each sample, wash the pestle in double-distilled water and dry it with a clean tissue. Change the pestle between different experimental conditions and tissues.
   4. Leave the samples on ice for 10 min after homogenization. Centrifuge the samples at >10,000 x g at 4 °C for 10 min.
   5. Transfer the supernatant to a new tube on ice without disturbing the pellet. The supernatant is the protein sample. Store the extracted protein at -80 °C or directly proceed to measure the protein concentration.
2. Quantification and normalization of protein concentration
   1. Measure the protein concentration using a bicinchoninic acid (BCA) assay. Prepare a BCA assay mix according to the manufacturer’s instructions in a 96-well optical plate using 200 µL of BCA mix per well.
      NOTE: Other quantification methods such as Lowry and Bradford assays can also be used to determine protein concentration as long as protein concentration is quantified consistently across experiments.
   2. Prepare bovine serum albumin (BSA) standards at increasing concentrations in triplicate and add 1 µL of each protein sample in duplicate. Incubate the 96-well plate in a preheated heat block at 60 °C for 10 min or longer if the protein concentration is expected to be low.
   3. After incubation, measure the absorption at 560 nm using a plate reader.
   4. 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. The R-squared value for the standard curve should be greater than or equal to 0.98 to accurately estimate sample protein concentration.
   5. Normalize the amount of protein by preparing dilutions of protein samples in sample buffer and ultrapure water. The total volume can be adjusted depending on the type of gel used. Loading 30 µg of protein per lane as a starting amount is recommended. Add a reducing agent such as dithiothreitol (DTT; final concentration 5 mM) or beta-mercaptoethanol (final concentration 200 mM) to each sample as required. Pipette undiluted beta-mercaptoethanol in a fume hood.
   6. Incubate the samples in a heat block at 70 °C for 10 min. Put the samples on ice, vortex, and spin down briefly to collect. Keep on ice until loading the gel.

2. Gel electrophoresis of protein samples

1. Device and gel setup
   1. Set up a precast 4–12% Bis-Tris gradient gel (see Table of Materials) in the gel electrophoresis chamber system. Rinse the gels using double-distilled water before use.
      NOTE: Depending on the size, interactions, and abundance of the protein of interest, gels with a different gradient, buffering agent, or well size and number can be used.
   2. Add 500 mL of 1x MES SDS running buffer diluted in double-distilled water per tank. Carefully remove the comb from the gels after adding the running buffer without disturbing the wells in the stacking gel.
2. Protein loading
   1. Load 3.5 µL of a protein standard into the well. Depending on the sample layout, loading a protein ladder on both sides of the gel can aid in more accurately estimating protein size. Use fine-tipped gel loading tips for more accurate sample loading.
   2. When using an internal standard for between-membrane normalization (see step 5 below and discussion), load an amount that is equal to the other samples into the first 3 wells next to the protein ladder.
   3. Load 30 µg of each sample in the remaining wells. Add 1x sample buffer to all empty wells.
3. Electrophoresis
   1. Assemble the gel tank after loading the samples. Run the samples through the stacking gel at 80 V for 10 min followed by 150 V for an additional 45-60 min.

3. Protein transfer

NOTE: Protein transfer in this protocol is performed using a commercially available semi-dry blotting system (see Table of Materials) for fast and consistent outcomes.

1. Prepare the protein transfer by pre-soaking filter paper in double-distilled water and ensuring the gel knife, plastic Pasteur pipette, blotting roller, and forceps are ready to use.
2. Open the transfer stack by carefully removing all wrapping foil. Remove the top from the bottom stack and set it aside. Quickly moisten the membrane on the bottom stack with several drops of electrophoresis running buffer (2-3 mL). Once the transfer stack is open, it is important to prevent the PVDF membrane from drying out.
3. After stopping the electrophoresis, open the pre-cast gel using the gel knife and cut off the stacking gel. Cut the gel around its edges to free it from the plastic cast. Keep the gel knife wet to prevent damage to the gel.
4. Assemble the transfer stack from bottom to top: bottom stack (containing the PVDF membrane), protein gel, and filter paper. Use the blotting roller to remove all air bubbles. Place the top stack on top of the filter paper and roll the stack again to remove air bubbles. Do not push too strongly as this may cause the gel to deform during protein transfer.
5. 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 it is aligned with the corresponding electrical contacts on the device. Close the lid, select, and start the appropriate program (20 V for 7 min is a recommended starting point).
6. When finished, leave the lid closed for 2 min to allow the stack to cool down and to prevent the membrane from drying out. Remove the transfer stack and cut the membrane to the gel size. Wash the cut membrane quickly with double-distilled water before continuing with the total protein stain.

4. Total protein staining

NOTE: Using fluorescent detection provides a substantial benefit over more traditional approaches (e.g., ECL detection), as the linear range and sensitivity can be much better controlled. Therefore, in steps 4 and 5, fluorescent TPS and fluorescent secondary antibodies are used (see Table of Materials).

1. Roll the membrane into a 50-mL tube with the protein side facing inwards. Because of the light sensitivity of the fluorescent TPS and secondary antibodies, all subsequent steps are carried out in the dark.
2. Add 5 mL of protein stain solution (see Table of Materials) and incubate on a roller for 5 min at room temperature. Because TPS and wash buffer contain methanol, carry these steps out in a fume hood.
3. Discard the staining solution and wash twice quickly with the 5 mL of wash solution (6.3% acetic acid in 30% methanol). Place the tube briefly back on the roller between the wash steps. Rinse the membrane briefly with ultrapure water before continuing.

5. Blocking, antibody incubation, and detection

1. Blocking the membrane: Add 3 mL of blocking buffer (see Table of Materials) to the 50 mL tube containing the membrane. Incubate the membrane on a roller for 30 min at room temperature. Depending on the choice of antibody, the type of blocking buffer used may require optimization.
2. Primary antibody incubation
   1. Discard the blocking buffer and replace it with the primary antibody at the appropriate, optimized concentration (Figure 1 and Figure 2: mouse-anti-SMN, at 1:1,000, diluted in blocking buffer).
      NOTE: Adequate optimization of primary antibodies should include confirmation that the antibody detects a protein product of the right size whilst showing no or minimal binding to other, unspecific protein products. If possible, test and compare multiple antibodies against the protein of interest.
   2. Incubate the membrane on a roller overnight at 4 °C. The next day, remove the antibody solution and wash it 6 times for 5 min with 1x PBS on a roller at room temperature (RT).
3. Secondary antibody incubation
   1. Prepare the specific secondary antibody at 1:5,000 against the host of the primary antibody in a 5 mL blocking buffer. Other secondary antibodies may require other dilutions or the use of alternative blocking buffers.
   2. Incubate the membrane with the secondary antibody solution on a roller for 1 h at RT. After incubation, wash the membrane three times 30 min with 1x PBS on a roller.
   3. Dry the membrane and keep the membrane protected from light using aluminum foil until detection. Membranes can be kept at 4 °C for long-term storage.
4. Image acquisition
   1. Log in to the computer attached to the scanner. Place the membrane on the scanner with the protein side facing down and select the scanning area in the software.
   2. Optimize the laser intensity for both (700 nm and 800 nm) channels, by confirming no saturation occurs. Acquire the images in both channels and export the images for further analysis (step 6).

6. Western blot analysis and quantification

NOTE: These recommendations are based on the freely available Image Studio software. However, comparable analyses can also be done using other software packages, such as ImageJ.

1. Import the file: Create a local workspace on the computer used for analysis. This generates a database of image files for acquired western blots. Import files obtained on the scanner and select the image for analysis.
2. TPS analysis
   1. Display the 700 nm channel to show the total protein staining result. An example of a TPS image is included in Figure 1 in which different tissues from neonatal (P5) (Figure 1A-B) and 10-week-old mice (Figure 1C-D) are compared directly. Similarly, Figure 2 shows an example of a direct comparison of brain tissue from mice of different ages.
   2. Select the Analysis tab from the top right corner and select Add Rectangle to define the area of interest for normalization (Figure 1B, D). Copy and paste the first rectangle area onto each individual sample to ensure the defined region is at the same size for all analyzed lanes.
   3. Copy the result from the Shapes tab in the bottom left corner of the software.
3. Quantification
   NOTE: The optimal approach for quantifying samples depends on the experimental design. We will here provide an illustrative example of the detection of the survival motor neuron protein (Smn; a key protein involved in the neuromuscular disease spinal muscular atrophy), which is known to decrease over time, and how the normalization of Smn signal intensity to TPS provides reliable estimates of protein expression development.
   1. Figure 2 shows a decrease of Smn expression with increasing age of the animal with TPS as a protein loading control. Repeat Steps 6.2.1-6.2.3 to quantify protein loading (Figure 2B). Repeat Steps 6.2.1-6.2.3 in the 800 nm channel (Figure 2A) to analyze the protein of interest.
   2. Copy the results from both TPS and protein of interest to a spreadsheet program. On the spreadsheet, first, normalize the protein loading by determining the highest TPS signal and dividing each TPS signal value by this value to obtain the normalized protein loading value.
   3. Divide the 800 nm signal value from each individual sample by its corresponding normalized protein value to calculate the relative protein expression ratio in different samples.
   4. After the first normalization, compare various time points or tissues to the average value of the internal standard to allow direct comparisons across different membranes and experiments.