western blot

For a detailed step-by-step Western blotting procedure see a recent article from our laboratory:

Detailed Western Blotting (Immunoblotting) Protocol (protocols.io)

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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.

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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.

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The transferred proteins retain their spatial arrangement, allowing for subsequent detection and analysis.

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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.

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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.

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Antibodies that are not highly specific often lead to inaccurate results.

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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.

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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.

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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.

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Advanced Steps

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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.

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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.

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Common Mistakes

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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.

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Not using a positive control when utilizing a new antibody. More antibodies are of “poor quality” compared to the number of “high quality” antibodies.