Western Blotting: Principles, Applications, and Experimental Techniques

What is Western Blot?

Western Blot (WB), also known as protein immunoblotting, is a conventional protein analysis technique commonly used to identify specific proteins and perform qualitative and semi-quantitative analysis of proteins.

The fundamental principle of WB experiments relies on the specific reaction between antigens and antibodies. Proteins of different molecular weights are first separated by denaturing gel electrophoresis using SDS-PAGE. Subsequently, they are transferred onto a solid support membrane, typically PVDF membrane. The membrane is then blocked using a blocking solution, often made from skim milk, to prevent non-specific binding, followed by incubation with a primary antibody, which is appropriately diluted. After the target protein and antibody have sufficiently bound, any excess unbound primary antibody is washed off using a TBST washing buffer. Next, a secondary antibody labeled with HRP (Horseradish Peroxidase) corresponding to the primary antibody is added. This forms a complex of protein-primary antibody-secondary antibody. Upon addition of a chemiluminescent substrate, fluorescence is generated at the location of the target protein. The bands corresponding to the target proteins can then be visualized using X-ray film or a chemiluminescence imager.

The typical workflow of a WB experiment includes protein extraction, protein quantification, gel casting, electrophoresis, membrane transfer, blocking, primary antibody incubation, primary antibody washing, secondary antibody incubation, secondary antibody washing, chemiluminescent detection, and data analysis.

What is a Western Blot Used For?

Western Blot, also known as immunoblotting, is a widely used analytical technique in molecular biology, biochemistry, and immunogenetics. Its primary purpose is to detect specific proteins in a sample of tissue homogenate or extract.

Western Blotting is used for several objectives in scientific research and medical testing. Firstly, it serves as an excellent means to detect and analyze proteins. Through this procedure, proteins can be identified in a complex mixture, even if they are in small concentration, providing useful data about their molecular weight and size. This is achieved by separating the proteins using gel electrophoresis before transferring them to a membrane and staining with antibodies.

Furthermore, Western Blot is an essential tool used in diagnostics and serology testing–most notably to confirm the presence of HIV, Lyme disease, Hepatitis B, and other virus antibodies in patient samples. The blotting technique allows for the identification and verification of antigen-specific antibodies, making it a reliable method for disease detection.

Additionally, Western Blot method is also useful in identifying modifications to proteins, such as phosphorylation, glycosylation, among others. Thus, contributing significantly to the understanding of molecular mechanisms and pathways for various biological processes and disease states.

Finally, it's important to note that while Western Blotting is a powerful tool, it also requires experiential skill and knowledge to interpret the results accurately. Therefore, a scientifically rigorous approach is vital when applying this technique.

What is the Difference Between ELISA and Western Blot?

Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting are both immunoassay techniques used to detect and quantify proteins, but they differ in several aspects:

• Principle: ELISA relies on the specific binding of an enzyme-conjugated antibody to an immobilized antigen, followed by enzymatic detection of the bound antibody. In contrast, Western Blotting involves the separation of proteins based on size by gel electrophoresis, followed by transfer onto a membrane and detection with specific antibodies.
• Sensitivity: Western Blotting generally offers higher sensitivity compared to ELISA, allowing for the detection of low-abundance proteins and subtle changes in protein expression levels.
• Specificity: Both techniques offer high specificity, but Western Blotting provides additional confirmation of protein identity through size-based separation and antibody recognition.
• Multiplexing: While ELISA can be adapted for multiplexed detection of multiple analytes in a single sample, Western Blotting is typically limited to the analysis of one or a few proteins per assay.

Preparation of Reagents for Western Blot Experiment

Before conducting a Western blot experiment, it is essential to prepare various reagents to ensure the smooth progress and accurate results of the assay. Below are the detailed instructions for preparing the necessary reagents:

Electrophoresis Buffer:

Prepare electrophoresis buffer by dissolving the appropriate amount of electrophoresis buffer powder in 1 liter of ultrapure water. Ensure thorough mixing by stirring on a magnetic stirrer until the powder is completely dissolved.

Transfer Buffer:

Dissolve the transfer buffer powder in 800 mL of ultrapure water. Add 200 mL of methanol to the solution and stir on a magnetic stirrer until completely dissolved.

TBST Buffer:

Prepare TBST buffer by dissolving TBS powder in 2 liters of ultrapure water. Add 1 mL of Tween-20 to the solution and stir on a magnetic stirrer until completely dissolved.

5% Non-Fat Dry Milk Solution:

Weigh 5 grams of non-fat dry milk powder and dissolve it in 100 mL of TBST buffer. Stir the solution on a magnetic stirrer for at least 30 minutes until the powder is completely dissolved. Store the solution at 4°C until use.

Complete Lysis Buffer:

Prepare complete lysis buffer by combining 1 mL of RIPA lysis buffer with 20 μL of cocktail, 10 μL of phosphatase inhibitor A, 10 μL of phosphatase inhibitor B, and 10 μL of PMSF. Add these components to the RIPA lysis buffer just before use.

These reagents are essential for various steps of the Western blot experiment, including protein electrophoresis, protein transfer to membranes, blocking, antibody incubation, and detection. Proper preparation and storage of these reagents are crucial for obtaining reliable and reproducible Western blot results.

Protein Extraction for Western Blotting

Total Protein Extraction from Tissues:

• Prepare the required number of sample tubes and place grinding beads inside them. Keep them on ice.
• Wash tissue blocks with pre-chilled PBS 2-3 times to remove blood contaminants.
• Place the tissue blocks on filter paper to remove excess PBS, then transfer them to corresponding homogenization tubes.
• Add approximately 10 times the volume of complete lysis buffer to the tissue homogenizer, ensuring balanced placement.
• Secure the lids and start homogenization.

Tips:

• For sesame-sized tissues, add 100-200 μL of complete lysis buffer; for bean-sized tissues, add 400-600 μL; for soybean-sized tissues, add 800-1000 μL.
• Parameters for the homogenizer: use 8-12 beads of 2mm zirconium oxide, set the frequency to 6.5 m/s, and homogenize for 30 seconds. Repeat homogenization if necessary.

Protein Extraction from Suspended Cells:

• Centrifuge the cells along with culture medium in a 15 mL tube at 4°C, 2000 rpm, for 5 minutes. Discard the supernatant.
• Resuspend the cells in 1 mL of PBS and transfer them to a 1.5 mL tube. Centrifuge again at 4°C, 2000 rpm, for 5 minutes. Discard the supernatant and retain the pellet.
• Repeat this washing step 2-3 times to remove residual culture medium.
• Based on the volume of cell pellet, add an appropriate amount of complete lysis buffer (e.g., for a pellet volume equivalent to a bean-sized volume, add about 200 μL of complete lysis buffer).
• Add grinding beads, then place the tubes in the homogenizer for protein extraction.
• After homogenization, centrifuge at 12000 rpm, 4°C, for 10 minutes, and collect the supernatant as the total protein solution.

Protein Extraction from Adherent Cells:

• Remove the culture medium from the cell culture dish or flask, and slowly add PBS along the side of the dish or flask.
• Tilt the dish or flask to remove residual PBS gently, avoiding cell detachment. Repeat washing 2-3 times, ensuring complete removal of residual PBS in the final wash.
• Add an appropriate volume of complete lysis buffer (e.g., for a six-well plate with confluent cells, add about 250 μL of complete lysis buffer).
• Gently shake the dish or flask to ensure full contact of cells with the lysis buffer, then scrape the cells with a cell scraper.
• Collect the lysate, add grinding beads, and homogenize the sample.
• After homogenization, centrifuge at 12000 rpm, 4°C, for 10 minutes, and collect the supernatant as the total protein solution.

The steps of western blotting (Roy et al., 2019).

Western Blotting Experimental Steps

Cleaning Glass Plates

Wash the glass plates of the vertical electrophoresis apparatus with detergent, rinse twice with water, and then rinse once with distilled water. Allow the plates to air dry or use a fan to dry them.

Preparation of Separating Gel and Stacking Gel

Depending on the target protein's molecular weight and the selected electrophoresis buffer, choose the appropriate concentration of SDS-PAGE separating gel. Prepare a 10% ammonium persulfate (AP) solution by dissolving 0.1 g of AP powder in 1 mL of distilled water. This solution can be stored at 4°C for 1-2 weeks or at -20°C for up to 3 months. Mix solutions A and B in equal proportions based on experimental requirements and add an appropriate amount of 10% AP solution to prepare the separating gel and stacking gel solutions. Adjust the amount of AP in the gel preparation according to the actual laboratory temperature; for example, reduce the amount in summer when gel polymerization is faster due to higher temperatures, and increase it in winter when gel polymerization is slower due to lower temperatures.

Gel Casting

Slowly pour the prepared separating gel solution into the space between the two glass plates until it reaches two-thirds to three-quarters of the chamber (based on experimental requirements). Then, use double-distilled water to evenly fill the chamber and create a seal (be cautious to avoid bubble formation). Allow the gel to polymerize for approximately 15-30 minutes, then pour out the excess water and use absorbent paper to clean any remaining liquid from the chamber. Fill the chamber with the prepared stacking gel solution (up to the top of the notches on the glass plate) and insert the comb. After waiting for approximately 15-30 minutes for complete gel polymerization, the gel casting process is complete.

Sample Loading and Electrophoresis

Once the stacking gel has solidified, remove the comb. If the sample wells are distorted, correct them using a sample-loading needle. Load the protein samples, with 5-10 μL of protein marker in the marker well and 30-50 μg of total protein in the sample wells. Place the gel cassette into the electrophoresis tank, fill the tank with electrophoresis buffer, and connect it to the power supply. Set the voltage to 60-90V initially, and then increase it to 120-150V after the samples have migrated into the separating gel. Terminate the electrophoresis when the bromophenol blue indicator is approximately 1 cm from the bottom of the gel. Ensure uniform sample loading to prevent uneven protein bands; if there are empty sample wells, add an equal volume of 1× loading buffer to prevent diffusion of neighboring protein samples. Take care to slowly add samples to avoid overflow, and ensure that the sample volume is smaller than the total volume of the sample wells. Fill the inner chamber of the electrophoresis tank with electrophoresis buffer and fill the outer chamber to one-fourth capacity. For 1.0 mm glass plates, a maximum of 20 μL can be loaded, while for 1.5 mm plates, up to 40 μL can be loaded. The voltage for the stacking gel should be adjusted based on temperature; for higher temperatures, lower the voltage (e.g., 75V in summer) and for lower temperatures, increase the voltage (e.g., 90V in winter), with a high voltage of 150V for the separating gel. Adjust the voltage and time according to the marker; if the marker bands separate, it indicates that the proteins have entered the separating gel. During hot weather, place the electrophoresis tank in ice water to cool it and prevent gel deformation due to high temperatures.

Reference

1. Roy, Jyoti, et al. "Small RNA proteome as disease biomarker: an incognito treasure of clinical utility." AGO-driven non-coding RNAs. Academic Press, 2019. 101-136.

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