Western blotting Protocol

Introduction

The Western blot is a biological technique that allows for the specific identification and characterization of proteins. In this technique, the proteins are assorted based on their molecular weight, and type, through gel electrophoresis. The results of the electrophoresis are then transferred to a polyvinylidene fluoride (PVDF) membrane producing a band for each protein. The membrane containing bands is then incubated with antibodies specific to the target protein. Western blotting facilitates the qualitative and quantitative assessment of protein expression in a variety of tissues and allows inferences about pre- and post-translational processes (e.g., phosphorylation or alternative splicing). The western blot is a powerful analytical tool used in molecular biology, immunogenetics and other molecular biology disciplines for protein characterization from a sample of tissue homogenate or extract.

The western blot is widely used in biochemistry for the qualitative evaluation of single proteins and protein-modifications (such as post-translational modifications). It is generally used as an analytical method to identify the presence of a specific single protein within a complex mixture. Semi-quantitative estimation of a protein can also be derived from the color intensity and the size of the band on the blot membrane. Besides, a dilution series of a purified protein of known concentrations can be used for precise estimation of protein concentration. The western blot is also used for medical diagnoses such as HIV-test and BSE-test.

Principle

Western blotting is based on the principle of immunochromatography where proteins are separated onto polyacrylamide gel according to their molecular weight. Proteins move within the gel onto a membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF). Proteins combine with membrane based on a hydrophobic interaction, thereby having a slight effect on protein activities. Then the isolated proteins are detected by conjugating them with primary antibody and secondary enzyme-labeled antibody and substrate.

Apparatus

Blotting equipment consists of the components required for protein transfer from the gel to a membrane and subsequent membrane processing. Blots require an apparatus, or blotter to transfer the gel to the membrane under dry, semi-dry, or wet conditions. Transfer protocol depends on the type of protein of interest, gel thickness, and the type of membrane. After protein transfer, the proteins must be labeled, stained, washed and detected. Automated equipment could be used to shake, block, hybridize, and wash blots, and recycle the primary antibody.

Protocol(Kim, 2017)

Polyacrylamide gel electrophoresis

1. Dilute 100 ml 10× [amazon link=”B01MU9TMB2″ link_icon=”amazon” /] using 900 ml of 18 MΩ-cm water to make it 1×.
2. Lock the gel into the gel apparatus before adding 300–350 ml of 1× running buffer in the central reservoir of the gel apparatus.
3. Load the sample into the respective lanes of the gel. Load 1 μL of Magicmark and 3 μL of BenchMark molecular markers.
4. Run the gel until the front reaches the bottom of the gel; it will take about 55 min.

Immunoblotting

1. Wet the protein-loaded membrane in [amazon link=”B0138M71OE” link_icon=”amazon” /] for 60 seconds. Transfer the membrane to a container with transfer buffer until needed.
2. Soak the sponges in [amazon link=”B0731SK3KK” link_icon=”amazon” /] Make sure there is no air bubble in the sponge.
3. Place the sponges into the blot module.
4. Soak the filter paper in the transfer buffer and place it in the blot modules.
5. Open the gel cassette. Cut the gel foot and lane dividers. Place the gel in the blot module.
6. Place the membrane in the blot module.
7. Wet the filter paper with [amazon link=”B0731SK3KK” link_icon=”amazon” /] (800 ml 18 MΩ-cm water, 200 ml methanol, 15.5 g Glycine, 3 g Tris Base) and place it in the blot module.
8. Close the blot module and insert it into the electrophoresis chamber.
9. Fill the blot module with transfer buffer. Fill the outer electrophoresis chamber to the half with distilled water.
10. Transfer the proteins to the membrane for 1.5 hours at 25 V.
11. Remove the membrane from the blot module, and place in >20 ml of I-BLOCK solution (1-liter [amazon link=”B00RKPGHUU” link_icon=”amazon” /], 2 g I-Block, 1 ml [amazon link=”B00I31RZWO” link_icon=”amazon” /]) for 1 hour at room temperature.

Detection

1. Prepare 10 ml of primary antibody (1:100) in 5% [amazon link=”B002L7TC5Y” link_icon=”amazon” /] solution (1:100) 20 minutes before use.
2. Incubate the membrane in primary antibody solution for 2 hours at room temperature.
3. Wash the membrane (four times) with >20 ml of I-BLOCK solution for 4 minutes each with gentle rocking.
4. Prepare 10 ml of secondary antibody (1:5000) in 5% NFDM solution at least 20 minutes before use.
5. Incubate the membrane loaded with the secondary antibody solution for 1 hour at room temperature.
6. Wash the membrane with >20 ml of I-BLOCK solution (twice), then with >20 ml [amazon link=”B00RKPGHUU” link_icon=”amazon” /] twice for 5 minutes each.
7. Mix 3 ml of ECL2 reagent during the last PBS wash.
8. Incubate the membrane in ECL2 reagent for 5 minutes in the dark.
9. Place the membrane in the development folder, and roll out any air bubbles.
10. Image the membrane.

Applications

Evaluating the protein components of the endocannabinoid system (Wager-Miller. & Mackie., 2016)

Western blotting facilitates the characterization and quantification of protein expression in the endocannabinoid system. The expression level of G protein-coupled receptors (GPCRs) is necessary to understand the function, distribution, and regulation of these receptors. Western blotting is a common approach for detecting proteins from complex biological systems. The western blotting was used to assess the protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, to detect the presence of type 1 cannabinoid (CB 1) receptors. The western blot presents the researchers with the expression level, distribution, and size details of the proteins of the endocannabinoid system.

Isolating and analyzing the extracellular vesicles (Kowal., Ovanesyan., Regev., & Church., 2017)

Extracellular vesicles (EVs) are responsible for intercellular communication and transport. The mammalian cells secrete heterogeneous vesicles of a variety of sizes; these vesicles can be categorized by the profiling of the proteins they contain through western blotting. It was found that the tetraspanins are a group of proteins containing four transmembrane domains. CD9, CD63, and CD81 are the transmembrane proteins commonly found in extracellular vesicles across cell types. Western blotting is a powerful technique used for the analysis of protein molecules and extracellular vesicles.

Antibody validation (Signore., Manganelli., & Hodge., 2017)

Antibody validation is usually done for translational research, particularly for biomarker discovery. Determination of the antibody specificity and protein biomarker identity is of critical importance for implementing the biomarkers in clinical studies, and the lack of such quality control tests may lead to unexpected or misleading results. In antibody validation, a single antibody is thoroughly assayed for its sensitivity and specificity. Antibody specificity is assessed by the presence of the band in a complex biological sample, at the expected molecular weight, on a Western blot. Antibodies used in the clinical research applications are validated using the standardized Western blot procedure in which control lysates containing the protein of interest, are probed with an antibody. The western blot is of critical importance in preclinical and clinical research to unveil the protein interactions and characteristic features.

Analysis of the antibody response to Campylobacter jejuni cellular antigens (Nachamkin. & Hart, 1985)

In the study, the western blot analysis was used to identify the antigenic components of Campylobacter jejuni that elicit antibody responses in humans suffering from campylobacter enteritis. Acute- and convalescent-phase sera were analyzed for humoral response against their homologous infecting strains and clinical isolates. In the acute-phase response, two to three antigens having molecular weights of 66,000 (p66), 43,000 to 46,000, and 12,000 (pl2) were detected in immunoblots. It was found that the convalescent-phase sera have a broader array of antibody binding to cell components. p66 was found to be the campylobacter flagellin, which is a major immunodominant component in almost all sera tested. The results showed that the antibody response against C. jejuni antigens is variable and out of the antigenic components the flagellin appeared to be the significant immunodominant component during infection.

Precautions

• Use the gel with a low acrylamide concentration when running high molecular weight proteins.
• To ensure a high resolution and efficient transfer, decrease the amount of bis-acrylamide.
• The high concentration of antibodies may coat the membrane with antibodies due to saturation, resulting in a uniform signal across the entire membrane so use a lower antibody concentration.
• Improper washing may cause excess antibody retaining, use wash buffers with Tween 20 to improve washing.
• Non-specific bands may develop because of the presence of the strong, anionic detergent SDS on the membrane, so wash the blot extensively after transferring the membrane.

Strengths and Limitations

• Western blot is a sensitive technique used as a useful early diagnostic tool, enabling the detection of even the slightest immunogenic response from a virus or bacteria in the sample.
• With western blotting, the sensitivity of the analysis can be increased even to observe ~10 x lower concentrations of the target proteins.
• The technique provides a much more powerful detection system that is essential for in vivo assays.
• Western blotting is a robust procedure for immunodetection of proteins, particularly which are of low abundance.
• In spite of its sensitivity and specificity, it could produce erroneous results. A false-positive develops when an antibody reacts with a non-intended protein.
• The cost of a western blot includes large expenditures for probed antibodies, skilled analysts, and the laboratory equipment.

References

1. Nachamkin., & Hart, A. M. (1985). Western blot analysis of the human antibody response to Campylobacter jejuni cellular antigens during gastrointestinal infection. J Clin Microbiol, 21(1), 33-38.
2. Wager-Miller., & Mackie., K. (2016). Western Blotting of the Endocannabinoid System. Methods Mol Biol, 1412, 247-54.
3. E. Kowal., D. Ter-Ovanesyan., A. Regev., & Church., M. G. (2017). Extracellular Vesicle Isolation and Analysis by Western Blotting. Methods Mol Biol, 1660, 143-152.
4. Kim, B. (2017). Western blot techniques. Methods Mol Biol, 1606, 133-139.
5. M. Signore., V. Manganelli., & Hodge., A. (2017). Antibody Validation by Western Blotting. Methods Mol Biol, 1606, 51-70.