Western blotting Protocol
WWestern Blotting (WB) is a widely used analytical technique in molecular biology and biochemistry for detecting specific proteins in a complex mixture of proteins extracted from cells or tissues. This method is essential for understanding protein expression, post-translational modifications, and protein-protein interactions. Western Blotting combines the resolving power of gel electrophoresis with the specificity of antibody-based detection, allowing researchers to identify a particular protein among a diverse array of proteins within a sample.
The Western Blot procedure involves several key steps: sample preparation, protein separation by size through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transfer of the proteins onto a membrane, blocking to prevent non-specific binding, incubation with primary and secondary antibodies, and finally, detection of the target protein.
Through its ability to detect specific proteins and analyze their expression levels, Western Blotting has become an indispensable tool in various research areas, including cell signaling, immunology, cancer research, and neurobiology. By providing insights into the molecular mechanisms underpinning biological processes and diseases, Western Blotting contributes significantly to advancing both basic and applied scientific research.
Western blotting (WB) Protocol
1. Sample Preparation
Materials:
• Sample (cells or tissue)
• Lysis buffer (e.g., RIPA buffer)
• Protease inhibitor cocktail
• Phosphatase inhibitor cocktail (for phosphorylated proteins)
• Loading buffer
• Dithiothreitol (DTT)
Steps:
1. Prepare Lysis Buffer:
• Prepare lysis buffer according to the manufacturer's instructions.
• Add protease and phosphatase inhibitors to the buffer to prevent protein degradation and dephosphorylation.
2. Cell Lysis:
• Suspension cells: Wash cells twice with PBS (phosphate-buffered saline) to remove any residual media. Resuspend the cell pellet in ice-cold lysis buffer.
• Adherent cells: Detach cells from the culture dish using a cell scraper or trypsinization. Collect cells and wash with PBS. Resuspend the cell pellet in ice-cold lysis buffer.
3. Lysis:
•Incubate the cell suspension in the lysis buffer on ice for 10 minutes. Gently rock or agitate the mixture to facilitate complete lysis.
4. Centrifugation:
•Centrifuge the lysate at 14,000–17,000 g for 5 minutes at 4°C to pellet cell debris.
•Collect the supernatant, which contains the protein lysate.
5. Protein Concentration Measurement:
•Measure the protein concentration using a Bradford or BCA assay according to the assay kit instructions.
6. Dilute Samples:
•Dilute the lysates to a final protein concentration of 1-2 mg/mL using loading buffer. Add DTT to reduce disulfide bonds if required.
7. Storage:
•Aliquot the protein samples to avoid repeated freeze-thaw cycles and store at -80°C until ready for SDS-PAGE.
rotein expression levels.
2. SDS-PAGE Electrophoresis
Materials:
•SDS-PAGE gel (e.g., Tris-Glycine system)
•Molecular weight markers
•Running buffer Electrophoresis
Steps:
1. Prepare Gel:
•Select the appropriate SDS-PAGE gel based on the size of the target protein. For example, use a 4-12% gradient gel for a range of protein sizes.
2. Load Samples and Run:
•Load 10-40 µg of protein sample into each well of the gel.
•Include molecular weight markers in one of the wells for size reference.
•Run electrophoresis at a constant voltage (typically 100-120V) until the bromophenol blue dye front reaches the bottom of the gel.
3. Protein Transfer
Materials:
•Transfer apparatus (wet or semi-dry)
•Membrane (nitrocellulose or PVDF)
•Transfer buffer
Steps:
1. Prepare Membrane:
•For PVDF membranes, pre-soak in methanol for a few seconds to activate. Then, rinse with water and equilibrate in transfer buffer.
•For nitrocellulose membranes, simply equilibrate in transfer buffer.
2. Assemble Transfer Apparatus:
•Set up the transfer stack according to the manufacturer's instructions, typically involving a "sandwich" of gel and membrane between filter papers and sponges.
•Ensure that there are no air bubbles between the gel and membrane.
3. Transfer:
•Run the transfer under recommended conditions (e.g., 30V for 1 hour for semi-dry transfer or 100V for 60-90 minutes for wet transfer). For overnight transfers, use a lower voltage (e.g., 10-30V).
4. Blocking and Antibody Incubation
Materials:
•Blocking buffer (e.g., 5% non-fat milk or BSA in TBST)
•Primary antibodies
•Secondary antibodies (HRP- or AP-conjugated)
•TBST (Tris-buffered saline with Tween 20)
Steps:
1. Block Membrane:
•Incubate the membrane in blocking buffer for 1 hour at room temperature with gentle shaking. This step helps to block non-specific binding sites.
2. Primary Antibody Incubation: •Dilute the primary antibody in blocking buffer according to the manufacturer's recommendation. •Incubate the membrane with the diluted primary antibody overnight at 4°C with gentle agitation.
3. Wash Membrane:
•Wash the membrane three times with TBST for 5 minutes each to remove unbound primary antibody.
4. Secondary Antibody Incubation:
•Dilute the HRP- or AP-conjugated secondary antibody in blocking buffer.
•Incubate the membrane with the secondary antibody for 1 hour at room temperature with gentle shaking.
5. Wash Membrane:
•Wash the membrane again with TBST three to six times for 5 minutes each to remove unbound secondary antibody.
5. Protein Detection
Materials:
•Chemiluminescent substrate (e.g., ECL) or other detection reagents
•Imaging system (e.g., X-ray film or chemiluminescent imaging system)
Steps:
1. Add Detection Reagent:
•Apply the appropriate chemiluminescent or colorimetric substrate to the membrane as per the manufacturer's instructions. Ensure even coverage of the membrane.
2. Image:
•Immediately capture the results using an imaging system. If using X-ray film, expose the film to the membrane in a darkroom. Alternatively, use a chemiluminescence imaging system to detect and capture the signal.
3. Analyze Results:
•Analyze the bands corresponding to the target protein and normalize against the loading control to quantify protein expression levels.
Algent WB Antibodies
Algent Bio offers a range of high-quality Western Blotting (WB) products designed to enhance the accuracy, sensitivity, and reproducibility of protein detection in your research. Here are some of the key advantages of using Algent Bio's WB solutions:
1. High Sensitivity and Specificity: Algent Bio provides highly specific primary and secondary antibodies, optimized to deliver strong, clean signals with minimal background noise. This ensures that only the target protein is detected, enhancing the accuracy of your results.
2.Comprehensive Product Range: Algent Bio offers a complete suite of Western Blotting reagents, including high-performance lysis buffers, protease and phosphatase inhibitors, blocking buffers, and detection substrates. This comprehensive range allows researchers to source all their Western Blotting needs from a single, reliable provider.
3. Superior Quality and Consistency: Our products undergo rigorous quality control to ensure batch-to-batch consistency, providing researchers with reliable results in every experiment. This reliability is crucial for long-term studies where reproducibility is key.
4. Optimized Protocols and Kits: Algent Bio provides optimized protocols and ready-to-use kits that simplify the Western Blotting workflow. Our protocols are designed to minimize hands-on time and reduce the risk of procedural errors, making them ideal for both novice and experienced researchers.
5 Enhanced Detection Systems: We offer advanced chemiluminescent and colorimetric substrates that provide high sensitivity and allow for the detection of low-abundance proteins. Our detection systems are compatible with a wide range of imaging equipment, providing flexibility in how you capture and analyze your results.
6. Technical Support and Expertise: Algent Bio's team of experienced scientists and technical support specialists is available to assist with troubleshooting and protocol optimization, ensuring you achieve the best possible outcomes in your research.
7. Cost-Effective Solutions: We offer competitive pricing on all our Western Blotting products without compromising on quality. Our solutions are designed to provide excellent value for money, helping to maximize your research budget.