Western Blot

Western blotting (WB) works by transferring protein samples separated by SDS-PAGE (based on molecular size) onto a solid support (e.g., nitrocellulose membrane), and then incubating the target protein with a specific antibody. Since the antibody only binds to the target protein, the unbound antibody is washed away, leaving only the antibody bound to the target protein. Finally, the bound antibody is detected by imaging, and the gray band corresponds to the protein amount, thus reflecting the protein expression status^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Protein Sample Preparation

1.1 Total Protein Extraction
(1) Solution Preparation
Melt the protein lysis buffer RIPA at room temperature, add the protein phosphatase inhibitor mixture (10X) to make the final concentration 1X, mix well, and immediately place on ice.
(2) Different Samples
For adherent cells: Wash 2-3 times with PBS pre-cooled to 4℃. Scrape the cells off with a cell scraper, or treat the cells with trypsin to loosen their adhesion and pipette them off. Centrifuge to collect the cells, and aspirate the supernatant as much as possible, leaving the cell pellet for later use.
For suspension cells: Wash 2-3 times with PBS, centrifuge to collect the cells, and aspirate the supernatant as much as possible, leaving the cell pellet for later use.
For tissue samples: Cut an appropriate amount of tissue and an appropriate amount of well-mixed protein lysis buffer and homogenize in a homogenizer (0.01 g tissue plus 50-100 μL of protein lysis buffer) until no tissue fragments are visible.
(3) Lysis
Add 100 μL of lysis buffer to cell samples at a cell count of 1×10⁶ and lyse on ice for 30 min (or lyse on ice for 5 min, then sonicate on ice for 20 s). Transfer the homogenate from tissue samples into 1.5 mL EP tubes and lyse on ice for 15 min.
(4) Extraction
Centrifuge at 12,000 rpm, 4℃ for 10 min, and immediately aspirate the supernatant into a pre-chilled 1.5 mL EP tube. This is the extracted cytoplasmic protein.
1.2 Bradford Method or BCA Protein Concentration Determination
Taking the Bradford method as an example
(1) Add 5 μL of gradient concentration protein standard solution to a 96-well plate;
(2) Add 5 μL of the sample to be tested to a 96-well plate. If the sample is less than 5 μL, add the sample stock solution or PBS to make up to 5 μL;
(3) Add 250 μL of G250 staining solution to each well and detect A595 using a microplate reader. The absorbance can be detected immediately or within 2 hours;
(4) Plot the enzyme standard curve: Plot the standard curve with the BSA standard protein concentration as the x-axis and the absorbance A595 at 595 nm as the y-axis;
(5) Calculate the protein concentration in the sample based on the standard curve and the volume of the sample to be tested.

2. Electrophoresis

2.1 Gel Preparation (Ignore this step if using pre-made gel plates)
(1) Prepare glass plates, ensuring the lower edges of both plates are horizontal and without gaps. When installing, the inner plate should be longer than the outer plate. Press the glass plates and the vertical tank gel preparation frame firmly by hand;
(2) Tilt the vertical tank gel preparation frame and insert it into the vertical tank gel preparation fixing frame. Note that to prevent gel deformation and leakage, do not press down excessively, and ensure it is securely fastened;
(3) Prepare 5 mL of separating gel (1 mm thickness) per plate, mix gently, and immediately spread it onto the plate;
(4) After spreading, flatten the surface of the separating gel with isopropanol;
(5) After solidification for 40 min, absorb the isopropanol;
(6) Prepare 1.5 mL of concentrated gel per piece, insert a comb of appropriate size, and ensure the operation is quick and level to avoid air bubbles;
(7) After the concentrated gel has solidified for 40 min, remove it from the gel preparation rack, clean the remaining gel on the gel plate with ddH2O, wrap it with plastic wrap, and use it immediately or store it at 4°C.
2.2 Sample Preparation
Add 4× loading buffer and mix well. Incubate at 100℃ for 10 min, then quickly cool in an ice bath.
2.3 Electrophoresis
(1) Based on the BCA assay results, load 20 μg-40 μg of the target protein per well and 5 μg-10 μg of the internal control protein per well.
(2) Use low-voltage constant-voltage electrophoresis (80 V) for the upper gel during electrophoresis, approximately 30 min.
(3) Use high-voltage constant-voltage electrophoresis (120 V) when bromophenol blue enters the lower gel. Stop electrophoresis when the bromophenol blue reaches near the bottom of the gel.

3. Transfer

Transfer steps:
(1) Prepare transfer buffer:
Prepare the transfer buffer in advance and pre-cool it to 4 ℃;
(2) Prepare PVDF membrane and filter paper:
Immerse the PVDF membrane in methanol for 30 s (from opaque to translucent), then rinse the membrane surface with ddH2O, and finally place the membrane and filter paper into the transfer buffer;
(3) Treat the gel:
Gently pry open the glass plate and cut off the stacking gel and the surrounding unwanted areas. Place the gel into the transfer buffer, ensuring the integrity of the gel;
(4) Prepare the transfer "sandwich":
Place the sandwich jacket with the black side down and the transparent side (or red side) up on a clean table. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the following order from bottom to top. Ensure there are no air bubbles between each layer. You can gently roll the top foam pad with a roller to remove air bubbles;
(5) Transfer:
Insert the jacket into the transfer electrophoresis core, ensuring the black plate faces the black plate. Then place the transfer electrophoresis core into the transfer tank, add enough transfer buffer, and ensure the jacket is completely submerged in the buffer. Set the transfer current to a constant current of 220 mA. Adjust the transfer time according to the protein size (generally 30 min for proteins below 30 kDa, 60-90 min for 30-70 kDa, and 90-180 min for 70-150 kDa);
(6) Post-transfer processing:
After transfer, remove the PVDF membrane, rinse the membrane surface with TBST, and proceed with subsequent steps such as blocking.

4. Blocking

At room temperature, place the transferred membrane on a shaker and block with 5% skim milk (prepared with TBST). For phosphorylated protein detection, use 5% BSA (prepared with TBST) buffer. Incubate at room temperature for 1-2 hours.
The main function of the blocking solution is to occupy non-target binding sites, reduce background signal, and thus improve the specificity and sensitivity of the detection. Commonly used blocking solutions: skim milk powder, bovine serum albumin (BSA).
Note: Skim milk powder is more effective when blocking non-phosphorylated proteins. This is because BSA contains only one protein with a molecular weight of 66 kDa, while milk powder contains multiple proteins, resulting in better blocking.

5. Antibody Incubation

(1) Incubation with primary antibody: Dilute the primary antibody (5% skim milk dissolved in TBST, 5% BSA dissolved in TBST for phosphorylated proteins), and incubate overnight at 4°C.
(2) Washing: Wash the membrane 3 times with TBST, 5 min each time.
(3) Incubation with secondary antibody: Generally use 5% skim milk dissolved in TBST, and 5% BSA dissolved in TBST for phosphorylated proteins, incubate at room temperature for 1-2 h.
(4) Washing: Wash the membrane 3 times with TBST, 1 time with TBS, 10 min each time.

6. ECL Development

(1) In a light-protected environment, prepare ECL chemiluminescence reagents A and B in a 1:1 ratio and mix thoroughly;
(2) Place the PVDF membrane on the stage of the chemiluminescence imaging instrument;
(3) Use a pipette to draw an appropriate amount of ECL mixture and drop it onto the PVDF membrane, ensuring that the working solution is evenly distributed across the entire imprint membrane;
(4) Push the instrument into the stage and set the exposure time for image acquisition (different exposure times can be set to acquire images, and the image with the best exposure effect can be selected).

1. Throughout the entire sample preparation process, the sample, buffer, and equipment should always be kept on ice, and the selected sample should be fresh.
2. When lysing cells/tissues, protease inhibitors should be added to the lysis buffer in advance to inhibit the degradation of cellular proteins by endogenous proteases.
3. When detecting phosphorylation, additional phosphatase inhibitors need to be added, preferably freshly added.
4. Avoid repeated freeze-thaw cycles for the extracted protein samples. For long-term storage, aliquots should be placed in a -80°C freezer. 5. For PVDF membranes, soaking in methanol for 30 seconds (make sure to completely wet the membrane!) is usually necessary to activate the positively charged groups on the membrane, making it easier for them to bind to negatively charged proteins. NC membranes need to be equilibrated in transfer buffer for a period of time.
6. After electrophoresis, carefully pry the gel off the glass plate to avoid damaging its integrity. The removed gel needs to be equilibrated in transfer buffer for a period of time to remove excess salts and other impurities.
7. During transfer, a "sandwich" structure should be constructed in the order of "filter paper-membrane-gel-filter paper". Ensure there are no air bubbles between each layer, otherwise it will affect the transfer efficiency.
8. To reduce non-specific binding, blocking buffer can be used as the antibody dilution solution for both primary and secondary antibodies.
9. The recommended incubation conditions for primary antibody are 4°C overnight and for secondary antibody are room temperature for 1 hour.
10. The membrane needs to be kept moist during washing to prevent it from drying out, which could lead to decreased antibody binding or protein denaturation.
11. The measured bands may differ from the expected molecular weight for reasons including:
(1) Post-translational modifications (such as phosphorylation and glycosylation) can increase the molecular weight of the protein.
(2) Splice variants and isoforms may produce proteins of different sizes from the same gene.
(3) Aggregation: Proteins may form polymers through strong interactions, resulting in bands at higher molecular weight positions. However, this phenomenon usually does not occur under reducing conditions.
(4) Relative charge: The amino acid composition affects the migration distance of the protein in the gel.