General Protocol for Western Blotting
Licia Miller Product Manager
Western blotting is a technique that uses specific antibodies to separate proteins and is widely used to analyze the expression of specific proteins in cell or tissue extracts. This protocol details the steps for performing western blotting of cell culture and tissue samples using both chemiluminescent and fluorescent detection .
Immunoassays use membranes made of nitrocellulose or PVDF (polyvinylidene fluoride). A gel is placed next to the membrane, and an electric current is applied , inducing the migration of proteins from the gel to the membrane. The membrane is then further treated with specific antibodies against the target and visualized using secondary antibodies and other detection reagents.
1. Sample preparation
The prepared lysate can be diluted in loading buffer in aliquots and stored frozen at -80°C until ready for use.
• Cell samples
Required Materials
- Lysis buffer (e.g. RIPA R301899)
- PBS
-Protease inhibitor cocktail (e.g. PMSF P408676)
- Phosphatase inhibitor cocktail (targeting phosphorylated proteins)
- Concentrated loading buffer
- BCA or Bradford assay kit
Experimental Steps
1. Add protease inhibitors to the lysis buffer and, if necessary, phosphatase inhibitors for phosphorylated proteins.
NOTE: Keep samples, buffers, and equipment on ice during the entire procedure.
2. Aspirate the medium from the culture; wash with 1× Wash cells with PBS; aspirate and resuspend the cell pellet in lysis buffer, typically about 1 mL of lysis buffer per 107 cells. Incubate at 4°C with shaking for 10 minutes. The suspension can be sonicated to break up the cells.
For suspension cells:
Wash cells twice with PBS by spinning (100 - 500 g, 5 min, 4°C) and resuspending the pellet.
Spin again (100-500 g, 5 min, 4°C) and resuspend in ice-cold lysis buffer.
It may be necessary to enzymatically or mechanically dissociate adherent cells before washing, spinning (100-500 g, 5 min, 4°C), and resuspending the pellet in lysis buffer.
3. Centrifuge the suspension at 14,000-17,000 g for 5 minutes at 4°C. Discard the pellet and transfer the supernatant to a fresh tube and place on ice.
4. Determine the protein concentration of the lysate using Bradford or BCA assay.
Tip: If the protein concentration is low at this stage and the protein is localized in the nucleus or mitochondria, consider fractionating the original sample to produce a more concentrated lysate.
5. Divide the lysate into several centrifuge tubes in equal amounts.
6. Dilute the aliquot in loading buffer. Add loading buffer to dilute the aliquot to a total protein concentration of approximately 1-2 mg/mL.
Note: Make sure the loading buffer contains DTT.
Diluting the lysate with loading buffer before storing in the refrigerator may make it more stable.
7. The samples are ready for direct use in subsequent experiments or can be stored at -80℃ until use.
• Tissue samples
Required Materials
-sample
- Lysis buffer (e.g. RIPA R301899)
- PBS
-Protease inhibitor cocktail (e.g. PMSF P408676)
- Phosphatase inhibitor cocktail (targeting phosphorylated proteins)
-Tubes filled with glass beads
-Automatic homogenizer
- Concentrated loading buffer
- BCA or Bradford assay kit
Experimental Steps
1. Add protease inhibitors to the lysis buffer and, if necessary, phosphatase inhibitors for phosphorylated proteins.
NOTE: Keep samples, buffers, and equipment on ice during the entire procedure.
2. Dissect the tissue on ice using clean tools . Place the dissected tissue and lysis buffer into a tube containing glass beads. Generally , add 1,200 μL of lysis buffer for every 200 mg of tissue.
NOTE: Do this as quickly as possible to prevent protease degradation.
If tissue samples are not homogenized immediately, they should be snap frozen in liquid nitrogen and stored at -80°C.
3. Use an automatic homogenizer to lyse the tissue suspension. Generally, the suspension is homogenized at 4°C for about 3 minutes and then incubated at 4°C for about 5 minutes.
Tip: You may need to pause the homogenization for 1 minute midway to avoid overheating the sample.
4. Centrifuge the suspension at 14,000-17,000 g for 5-10 minutes at 4°C to pellet insoluble material.
5. Transfer the supernatant to a new tube and place on ice.
6. Measure the protein concentration of the lysate using Bradford or BCA assay.
NOTE: If the protein concentration is low at this stage and the protein is localized in the nucleus or mitochondria, one may consider fractionating the original sample to produce a more concentrated lysate.
7. Divide equal volumes of the lysate into several centrifuge tubes.
8. Dilute the aliquot in loading buffer . Add loading buffer to dilute the aliquot to a total protein concentration of approximately 1-2 mg/mL.
Note: Make sure the loading buffer contains DTT.
Tip: Diluting the lysate with loading buffer before storing in the refrigerator may make it more stable.
9. The samples are ready for direct use in subsequent experiments or can be stored at -80° until use.
2. Sample loading and gel running
The gel is immersed in a buffer, a protein sample is added, and then an electric current is applied to the gel to cause the protein to migrate from one end of the gel (the negative electrode) to the other end (the positive electrode). Proteins of different molecular weights migrate at different speeds, and smaller proteins move faster in the gel and appear further down , thereby achieving the separation of proteins of different sizes.
Required Materials
- SDS-PAGE gel (Tris-Glycine, Bis-Tris or Tris Acetate gel)
- Gel electrophoresis instrument
- SDS
- Electrophoresis buffer (e.g. Tris-Glycine, MES, MOPS, Tris-Acetate)
Experimental Steps
1. Select the appropriate SDS-PAGE gel according to the size of the target protein and set up the running device. Place the gel in the running apparatus and fill it with electrophoresis buffer so that the gel is completely submerged.
Table 1: Recommended gradient gel chemistries for different protein sizes. Our laboratory uses gradient gels, but gels with a fixed acrylamide concentration can also be used.
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Table 2: Recommended resolving gel concentrations and loading buffers for proteins of different sizes . Generally, 10 - 15% resolving gel is a good starting point.
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Tip: The larger the protein, the lower the percentage of acrylamide in the gel. This will form less dense aggregates, making it easier for the protein to pass through.
Note: When running the gel, make sure the positive and negative electrodes of the electrophoresis instrument are inserted correctly.
2. Thaw the lysate and boil the lysate at 95-100°C for 10 minutes. Cool on ice.
3.Load an equal amount of protein from each sample onto the gel.The recommended loading amount is generally 10 - 40 µg of lysate protein and 10-500 ng of purified protein.
NOTE: Make sure the wells are straight before adding sample.
Do not overload the wells; this may cause sample to spill into adjacent wells.
At the same time, do not let the pipette tip touch the bottom of the well, which can easily cause the ribbon to distort.
4. Set the running time and voltage and perform gel electrophoresis.
Note: Ideal run times and voltages will vary by manufacturer, gel composition, and target protein. Larger proteins should be run longer at higher voltages.
5. After running, use a gel knife to carefully pry open the device and remove the gel.
3. Transfer from gel to membrane
After electrophoresis, the proteins are transferred to a membrane for antibody incubation. The membrane can be made of nitrocellulose or polyvinylidene fluoride (PVDF).
• Semi-dry transfer
Semi-dry transfer requires additional equipment, but transfer times are shorter and setup is easier.
Required Materials
- SDS-PAGE gel
-Transfer device
- Washing buffer (e.g. TBST)
- Membrane (nitrocellulose or PVDF)
- Methanol (if using PVDF)
Experimental Steps
1. If using PVDF membrane, soak the membrane in methanol . Then soak in transfer buffer at 4°C for 10 minutes.
Tip: This step is essential for PVDF membranes, as PVDF is naturally hydrophobic and needs to be activated with methanol to allow buffer to pass through efficiently. However, it is not necessary for nitrocellulose membranes.
2. Assemble the SDS-PAGE gel and membrane into the transfer box in the correct position. The gel should be closest to the negative electrode. Membrane should be closest to the positive electrode. Press down on the paper stack with a small roller to remove any air bubbles.
Note: Make sure the filter paper is cut to the same size as the membrane.
3. Set the running time and voltage to transfer the membrane.
4. Remove the gel and membrane from the transfer apparatus.
• Wet transfer
Wet transfer is performed in a tank. It does not require as much additional equipment as semi-dry transfer, but the transfer time is generally longer.
Required Materials
- SDS-PAGE gel
- Transfer device
- Transfer buffer (e.g. Tris-Glycine)
- Washing buffer (e.g. TBST)
- Membrane (Nitrocellulose or PVDF - Examples: ab133411, ab133412, ab133413)
- Methanol (if using PVDF)
Experimental Steps
1. If using PVDF, soak the membrane in methanol. Then soak in transfer buffer at 4°C for 10 minutes.
Tip: This step is essential for PVDF membranes, as PVDF is naturally hydrophobic and needs to be activated with methanol to allow buffer to pass through efficiently. However, it is not necessary for nitrocellulose membranes.
2. Assemble the SDS-PAGE gel and membrane in the transfer apparatus in the correct position . The gel should be closest to the negative electrode. The membrane should be closest to the positive electrode. Press down on the paper stack with a small roller to eliminate all bubbles.
NOTE: Use plastic tweezers to handle the membrane.
Make sure the filter paper is cut to the same size as the membrane.
3. Set the appropriate running time and voltage according to your needs.
4 Remove the gel and membrane from the transfer apparatus.
4. Membrane Blocking and Antibody Incubation
Use the following steps for antibody incubation. If using a loading control antibody in a chemiluminescent western blot, the following staining steps can be repeated on the same membrane after stripping.
In fluorescent western blotting experiments, multiple sets of antibodies can be incubated with the membrane simultaneously as follows. Loading control antibodies and detection antibodies can be run on the same membrane without stripping.
• For conjugated primary antibodies
Required Materials
- Blocking buffer (e.g. 3 - 5% milk or BSA in TBST, or a non-mammalian protein buffer)
- Washing buffer (e.g. TBST)
- membrane
- Conjugated primary antibody
Experimental Steps
1. After transfer, wash the nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature. (Optional )
2. Place the membrane in 25 ml of blocking buffer and incubate at room temperature for 1 hour.
Tip: Blocking buffer contains milk or BSA (3-5% in TBST).
Generally, BSA gives clearer results because there are fewer proteins in BSA that the antibody can cross-react with. Some antibodies work better with milk because it contains a wider variety of blocking proteins.
3. Wash three times with 15 ml TBST, 5 minutes each time.
4. Incubate the membrane and primary antibody (at the appropriate dilution and diluent recommended in the product datasheet) in 10 ml primary antibody dilution buffer at 4°C overnight with gentle agitation or at room temperature for 1 hour.
Note: Make sure the primary antibody dilution completely covers the membrane. Incubation time may need to be optimized depending on the specific experiment.
5. Wash the membrane three times with 15 ml TBST wash buffer for 5 minutes each.
• For unconjugated primary antibodies
Required Materials
- Blocking buffer (e.g. 3-5% milk or BSA in TBST, or a non-mammalian protein buffer)
- Washing buffer (e.g. TBST)
- membrane
- Primary Antibody
- Conjugated secondary antibody
Experimental Steps
1. After transfer, wash the nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature. (Optional)
2. Place the membrane in 25 ml of blocking buffer and incubate at room temperature for 1 hour.
Tip: Blocking buffer contains milk or BSA (3-5% in TBST).
Generally, BSA gives clearer results because there are fewer proteins in BSA that the antibody can cross-react with. Some antibodies work better with milk because it contains a wider variety of blocking proteins.
3. Use 15 Wash three times with 1 ml TBST for 5 min each.
4. Incubate the membrane and primary antibody (at the appropriate dilution and diluent recommended in the product datasheet) in 10 ml of primary antibody dilution buffer at 4°C overnight with gentle agitation or at room temperature for 1 hour.
Note: Make sure the primary antibody dilution completely covers the membrane. Incubation time may need to be optimized depending on the specific experiment.
5. Wash the membrane three times with 15 ml TBST wash buffer for 5 minutes each.
6. Add 10 ml of secondary antibody diluted in blocking buffer and incubate with gentle shaking at room temperature for 1 hour.
7. Wash the membrane three times with 15 ml TBST wash buffer for 5 minutes each.
5. Protein Detection
In chemiluminescent detection, the first step is to incubate the blot in a chemiluminescent substrate solution so that light will be emitted when the HRP-conjugated antibody is present. These bands of light on the blot correspond to antibody binding. Chemiluminescent blots have traditionally been imaged using X-ray film-based techniques, but these techniques are being replaced by benchtop charge-coupled device (CCD) imagers, which provide higher quality images.
For specific experimental operations, follow the instructions of the purchased instrument and chemiluminescence colorimetric kit and optimize according to your own experimental needs when necessary.
For more product details, please visit Aladdin Scientific website.