The Western Blotting Workflow: Step-by-Step by CLYTE.tech
1. Sample Preparation:
Lysis: The first step is to extract proteins from cells or tissues. This is typically achieved using lysis buffers containing detergents (like SDS or Triton X-100) to solubilize proteins and protease/phosphatase inhibitors to prevent protein degradation or modification. The choice of buffer depends on the protein's cellular location (cytoplasmic, nuclear, membrane-bound) and the experimental goal.
Quantification: Accurately determining the total protein concentration in each lysate is essential for comparing protein levels between samples. Common methods include the Bradford or BCA protein assays. Equal amounts of total protein are then loaded for each sample in the subsequent step.
2. Gel Electrophoresis (SDS-PAGE):
Denaturation: Samples are typically mixed with a loading buffer containing SDS (Sodium Dodecyl Sulfate) and a reducing agent (like DTT or β-mercaptoethanol) and heated. SDS coats proteins with a negative charge, masking their intrinsic charge, while the reducing agent breaks disulfide bonds, linearizing the proteins.
Separation: The denatured protein samples are loaded into the wells of a polyacrylamide gel (PAGE). When an electric field is applied, the negatively charged proteins migrate through the gel matrix towards the positive electrode. The gel acts as a sieve, separating proteins primarily based on their molecular weight – smaller proteins move faster and further down the gel.
3. Protein Transfer (Blotting) The proteins separated within the fragile gel must be transferred to a more durable membrane support (typically Nitrocellulose or PVDF) for probing. The membrane binds the proteins, creating a mirror image of the gel's protein pattern. Researchers commonly choose between two main methods: Wet (Tank) Transfer or Semi-Dry Transfer.
4. Blocking To prevent antibodies from binding non-specifically to the surface of the membrane, which creates unwanted background signal, the membrane must be blocked. This is done by incubating the membrane in a blocking solution, commonly 3-5% Bovine Serum Albumin (BSA) or Non-Fat Dry Milk, dissolved in a wash buffer like TBST (Tris-Buffered Saline with 0.1% Tween-20) or PBST.
5. Antibody Incubation:
Primary Antibody: The blocked membrane is incubated with a primary antibody solution (diluted in blocking buffer or TBST/PBST). The primary antibody is specifically chosen to recognize and bind to the target protein of interest. Incubation typically occurs for several hours at room temperature or overnight at 4°C with gentle agitation.
Washing: After primary antibody incubation, the membrane is washed multiple times with washing buffer (e.g., TBST/PBST) to remove unbound primary antibody. Thorough washing is critical to minimize background noise.
Secondary Antibody: The membrane is then incubated with a secondary antibody solution. This antibody is directed against the host species of the primary antibody (e.g., anti-rabbit IgG if the primary was raised in a rabbit). Importantly, the secondary antibody is conjugated to an enzyme (like Horseradish Peroxidase, HRP) or a fluorescent dye, which provides the means for detection.
Washing: Another series of washes removes unbound secondary antibody.
6. Detection and Imaging Visualizing the protein band relies on the signal generated by the label on the secondary antibody. The three primary approaches are Chemiluminescent, Fluorescent, and Colorimetric.
7. Analysis:
Interpretation: The presence and intensity of the band at the expected molecular weight confirm the detection of the target protein. The band intensity is generally proportional to the amount of target protein, allowing for semi-quantitative or quantitative analysis (often relative to a loading control – a housekeeping protein like actin or GAPDH that should be consistently expressed).
1. Sample Preparation: Lysis: The first step is to extract proteins from cells or tissues. This is typically achieved using lysis buffers containing detergents (like SDS or Triton X-100) to solubilize proteins and protease/phosphatase inhibitors to prevent protein degradation or modification. The choice of buffer depends on the protein's cellular location (cytoplasmic, nuclear, membrane-bound) and the experimental goal. Quantification: Accurately determining the total protein concentration in each lysate is essential for comparing protein levels between samples. Common methods include the Bradford or BCA protein assays. Equal amounts of total protein are then loaded for each sample in the subsequent step.
2. Gel Electrophoresis (SDS-PAGE): Denaturation: Samples are typically mixed with a loading buffer containing SDS (Sodium Dodecyl Sulfate) and a reducing agent (like DTT or β-mercaptoethanol) and heated. SDS coats proteins with a negative charge, masking their intrinsic charge, while the reducing agent breaks disulfide bonds, linearizing the proteins. Separation: The denatured protein samples are loaded into the wells of a polyacrylamide gel (PAGE). When an electric field is applied, the negatively charged proteins migrate through the gel matrix towards the positive electrode. The gel acts as a sieve, separating proteins primarily based on their molecular weight – smaller proteins move faster and further down the gel.
3. Protein Transfer (Blotting) The proteins separated within the fragile gel must be transferred to a more durable membrane support (typically Nitrocellulose or PVDF) for probing. The membrane binds the proteins, creating a mirror image of the gel's protein pattern. Researchers commonly choose between two main methods: Wet (Tank) Transfer or Semi-Dry Transfer.
4. Blocking To prevent antibodies from binding non-specifically to the surface of the membrane, which creates unwanted background signal, the membrane must be blocked. This is done by incubating the membrane in a blocking solution, commonly 3-5% Bovine Serum Albumin (BSA) or Non-Fat Dry Milk, dissolved in a wash buffer like TBST (Tris-Buffered Saline with 0.1% Tween-20) or PBST.
5. Antibody Incubation: Primary Antibody: The blocked membrane is incubated with a primary antibody solution (diluted in blocking buffer or TBST/PBST). The primary antibody is specifically chosen to recognize and bind to the target protein of interest. Incubation typically occurs for several hours at room temperature or overnight at 4°C with gentle agitation. Washing: After primary antibody incubation, the membrane is washed multiple times with washing buffer (e.g., TBST/PBST) to remove unbound primary antibody. Thorough washing is critical to minimize background noise. Secondary Antibody: The membrane is then incubated with a secondary antibody solution. This antibody is directed against the host species of the primary antibody (e.g., anti-rabbit IgG if the primary was raised in a rabbit). Importantly, the secondary antibody is conjugated to an enzyme (like Horseradish Peroxidase, HRP) or a fluorescent dye, which provides the means for detection. Washing: Another series of washes removes unbound secondary antibody.
6. Detection and Imaging Visualizing the protein band relies on the signal generated by the label on the secondary antibody. The three primary approaches are Chemiluminescent, Fluorescent, and Colorimetric.
7. Analysis: Interpretation: The presence and intensity of the band at the expected molecular weight confirm the detection of the target protein. The band intensity is generally proportional to the amount of target protein, allowing for semi-quantitative or quantitative analysis (often relative to a loading control – a housekeeping protein like actin or GAPDH that should be consistently expressed).