western blot
Theory, Procedure, Uses
Aldrin V. Gomes, Ph.D.
THEORY
A Western blot is the result obtained from the Western blotting procedure, and was first introduced by Towbin, et al. in 1979. Since then, hundreds of thousands of publications have included western blotting data. Probably a better name for Western blotting is immunoblotting, since it involves detection of proteins by antibodies (immune proteins). In the context of molecular biology and biochemistry, the term "blotting" refers to the transfer of biological molecules, typically proteins or nucleic acids, from a gel matrix onto a solid support medium, such as a membrane. This transfer is an essential step in various laboratory techniques used for the detection, analysis, and characterization of specific molecules within complex biological samples. There are several types of blotting techniques, each tailored to detect different types of molecules:
1. Southern Blotting: Used to detect specific DNA sequences within a sample. After gel electrophoresis, DNA fragments are transferred to a membrane and then hybridized with a labeled DNA probe that is complementary to the target sequence.
2. Northern Blotting: Similar to Southern blotting, but used for the detection of specific RNA molecules. RNA samples are separated by gel electrophoresis, transferred to a membrane, and hybridized with a labeled RNA probe.
3. Western Blotting (Immunoblotting): Employed for the detection and analysis of specific proteins within a sample. After protein separation by gel electrophoresis, proteins are transferred to a membrane and probed with specific antibodies that recognize the target protein.
4. Eastern Blotting: Used for the detection and analysis of post-translational modifications (PTMs) of proteins, such as glycosylation. After protein separation by gel electrophoresis, proteins are transferred to a membrane and probed with specific antibodies or lectins that recognize the PTMs.
In all types of blotting, the transfer process typically involves applying an electric current or pressure to facilitate the movement of molecules from the gel onto the membrane. The transferred molecules retain their spatial arrangement, allowing for subsequent detection and analysis using various labeling and detection methods.
Western blotting several key steps: first, the separation of proteins by size through gel electrophoresis; second, the transfer of these proteins from the gel to a solid support membrane; third, the binding of specific antibodies to the target proteins on the membrane; and finally, the detection and visualization of these proteins using various labeling and detection methods. This technique allows researchers to analyze protein expression levels, post-translational modifications, and interactions, providing valuable insights into cellular processes and disease mechanisms. Additionally, caution is advised when normalizing results, with recent evidence suggesting potential limitations in using housekeeping proteins for this purpose, recommending total protein normalization instead. Overall, Western blotting plays a critical role in elucidating the complexities of protein biology and remains an indispensable tool in biomedical research.
Procedure
For a detailed step-by-step Western blotting procedure see a recent article from our laboratory:
Detailed Western Blotting (Immunoblotting) Protocol (protocols.io)
Sample Preparation: The first crucial step in Western blotting is sample preparation. Biological samples containing proteins of interest, typically obtained from tissues or cell lysates, must be homogenized, and denatured to release their protein content. Denaturation, often achieved by heating the samples in a buffer containing reducing agents like β-mercaptoethanol, disrupts protein structures, ensuring uniform migration during subsequent steps.
Polyacrylamide Gel Electrophoresis (PAGE): Following sample preparation, the denatured proteins are separated based on their size through polyacrylamide gel electrophoresis (PAGE). In this step, denatured protein samples are loaded onto a gel matrix and subjected to an electric field. This technique relies on the differential migration of proteins in an electric field through a porous gel matrix. The gel's pore size dictates the resolution of protein separation, with smaller pores facilitating the separation of low molecular weight proteins. The separated proteins form distinct bands within the gel, representing individual components of the sample.
The transferred proteins retain their spatial arrangement, allowing for subsequent detection and analysis.
Transfer to Membrane: Once protein separation is complete, the next step involves transferring the proteins from the gel onto a solid support membrane, typically made of nitrocellulose or polyvinylidene difluoride (PVDF). This transfer, known as "blotting," is achieved through electroblotting, where an electric field is applied to facilitate the migration of proteins from the gel to the membrane. This transfer preserves the spatial arrangement of proteins, allowing for subsequent detection and analysis.
Blocking and Antibody Incubation: To prevent non-specific binding and enhance detection sensitivity, the transferred membrane is incubated in a blocking solution, such as bovine serum albumin (BSA) or non-fat dry milk. Blocking agents saturate unoccupied binding sites on the membrane, minimizing background signal during antibody incubation. Following blocking, the membrane is probed with specific primary antibodies targeting the protein of interest. These antibodies are generally highly specific and typically raised against epitopes unique to the target protein. Following primary antibody incubation, the membrane is washed to remove unbound antibodies.
Antibodies that are not highly specific often lead to inaccurate results.
Detection and Visualization: After primary antibody incubation, the membrane is washed to remove unbound antibodies, followed by incubation with secondary antibodies conjugated to detection molecules such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase) or fluorophores. The secondary antibodies recognize and bind to the primary antibodies, amplifying the signal for enhanced detection. Finally, the protein bands of interest are visualized using various detection methods, including chemiluminescence, fluorescence, or chromogenic substrates. The intensity of the detected bands correlates with the abundance of the target protein in the original sample.
Data Analysis and Interpretation: The final step in Western blotting involves data analysis and interpretation. The intensity and size of protein bands are quantified using image analysis software, allowing for comparative analysis between samples and conditions. Normalization against internal controls or loading controls helps correct variations in protein loading and transfer efficiency. By comparing protein expression levels across different experimental conditions, researchers can gain insights into cellular processes, biomarker identification, and disease mechanisms.
Approach with caution when normalizing Western blotting results using housekeeping proteins, as emerging evidence, indicates their non-linearity with typical total protein amounts loaded on gels. Opting for total protein normalization is strongly advised.
Advanced Steps
After removing the nitrocellulose membrane from the transfer cassette quickly rinse once in distilled water, then let the membrane dry. Use ponceau S staining for 2 minutes and rinse gently with water until the protein bands are clear. Image the picture to be used for protein normalization. Remove the ponceau stain by washing it in water or TBS.
The most used blocking buffer is 5% non-fat milk in TBS. However, we have found that 3% non-fat milk works much better with respect to the intensity of the bands detected.
Common Mistakes
Using non-fat milk when doing Western blots of phosphor-specific antibodies. Non-fat milk contains casein, a phosphorylated protein that can cause a high background on Western blots.
Not using a positive control when utilizing a new antibody. More antibodies are of “poor quality” compared to the number of “high quality” antibodies.
WESTERN BLOT USES
Western blotting is a versatile technique with numerous applications across various scientific disciplines. Here are twenty common uses:
1. Protein Expression Analysis: Western blotting allows researchers to quantify and compare the expression levels of specific proteins in different biological samples, providing insights into cellular processes and protein function.
2. Biomarker Detection: Western blotting can be used to identify and validate biomarkers associated with diseases such as cancer, neurological disorders, and autoimmune diseases, aiding in diagnosis and prognosis.
3. Detection of Post-Translational Modifications: Western blotting enables the detection of post-translational modifications (PTMs) such as phosphorylation, glycosylation, and ubiquitination, providing insights into protein regulation and signaling pathways.
4. Drug Development and Validation: Western blotting is employed in drug discovery and development to assess the efficacy and specificity of potential therapeutic agents, as well as to evaluate their effects on target proteins and signaling pathways.
5. Viral and Bacterial Protein Analysis: Western blotting is used to detect and characterize viral and bacterial proteins, aiding in the study of infectious diseases, vaccine development, and antimicrobial drug discovery.
6. Stem Cell Research: Western blotting is utilized in stem cell research to characterize the expression of stem cell markers and differentiation markers, as well as to monitor changes in protein expression during cellular reprogramming and differentiation.
7. Immunology and Antibody Characterization: Western blotting is employed to analyze antibody specificity, affinity, and cross-reactivity, as well as to confirm the presence of specific antigens in immunological assays.
8. Protein-Protein Interactions: Western blotting can be used to study protein-protein interactions by assessing the co-immunoprecipitation of interacting proteins or the formation of protein complexes under different experimental conditions.
9. Quality Control in Biotechnology: Western blotting is utilized in biotechnology and pharmaceutical industries for quality control purposes, including the assessment of protein purity, identity, and integrity in recombinant protein production.
10. Forensic Analysis: Western blotting can be applied in forensic science to detect and analyze protein markers for the identification of bodily fluids, tissue samples, and other biological evidence in criminal investigations.
11. Cancer Biomarker Profiling: Profiling protein biomarkers associated with different cancer types for early detection and personalized treatment strategies.
12. Signal Transduction Studies: Investigating signaling pathways by analyzing the activation status of key proteins, kinases, and transcription factors.
13. Immunoprecipitation Validation: Confirming the specificity of immunoprecipitation assays by Western blot analysis of precipitated proteins.
14. Transgene Expression Analysis: Assessing the expression of transgenes in genetically modified organisms (GMOs) and cell lines.
15. Vaccine Development: Evaluating the immunogenicity of vaccine candidates by detecting antigen-specific antibody responses in serum samples.
16. Cell Cycle Analysis: Monitoring changes in the expression of cell cycle regulatory proteins during cell proliferation and differentiation.
17. Apoptosis Detection: Assessing protein markers of apoptosis and programmed cell death in response to various stimuli or treatments.
18. Cellular Stress Response: Analyzing the expression of stress-related proteins such as heat shock proteins (HSPs) and transcription factors under conditions of cellular stress.
19. Host-Pathogen Interactions: Investigating protein expression changes in host cells during infection with pathogens such as bacteria, viruses, and parasites.
20. Diagnostic Assays: Developing Western blot-based diagnostic tests for infectious diseases, genetic disorders, and other medical conditions.
These are just a few examples of the diverse applications of Western blotting, highlighting its importance in basic research, clinical diagnostics, and applied sciences.
Conclusion
Western blotting is a powerful and versatile technique for protein analysis, offering valuable insights into cellular processes and disease mechanisms. By following the basic steps outlined above, researchers can effectively perform Western blot experiments, unraveling the complexities of protein expression and function. As technology advances and methodologies evolve, Western blotting continues to be a cornerstone technique in molecular biology, driving innovation and discovery in diverse fields of research.