13.12: Lab Technique - Western Blotting

Analyzing Proteins Through Western Blotting

The Western blot is a widely used laboratory technique that uses antibodies to identify a specific protein within a sample, quantify the expression of a protein as a result of experimental treatment, and detect post-translational modifications, such as phosphorylation or glycosylation. Western blotting (also called immunoblotting) begins with the separation of proteins through SDS-PAGE. Because the acrylamide gel is delicate, the proteins are electrically transferred or "blotted" from the gel onto a positively-charged matrix to capture the proteins (usually nitrocellulose paper). This produces an exact replica of the proteins on the substrate. This "copy" is then incubated with antibodies to detect the target protein. In a direct western blot, a single antibody, called a primary antibody is used to detect a specific region of a target protein. This primary antibody is coupled to some sort of marker (e.g. fluorescent, chemiluminescent). When the marker is detected, the protein is identified. In the more common indirect western blot, a primary antibody and a secondary antibody is used. The primary antibody is detected using the secondary antibody that is coupled to a chemiluminescent or fluorescent marker (Figure \(\PageIndex{1}\)) The indirect method allows for amplification of the signal as multiple labeled secondary antibodies can bind a primary antibody. The most common type of marker is a chemiluminescent marker, such as horseradish peroxidase (HRP), that develops a substrate to produce a luminescent signal. This signal can be detected using a documentation system or light-sensitive film. Once visualized, the luminescent protein band can be compared to protein markers of known size to confirm its size and compared to reference standards to quantify its expression level.

Key Components of the Western Blot

• SDS-PAGE gel: can be pre-cast or hand cast. For more information, see Laboratory Technique: SDS-PAGE
• Transfer apparatus: used to transfer the proteins in the acrylamide gel to a secondary matrix support
  • most common method is electrophoretic
  • can be done under wet, semi-dry or dry conditions
  • wet transfer:
    • most common method
    • requires use of a transfer tank, transfer cassette, and nitrocellulose membrane
    • transferred using a Transfer Buffer containing methanol
    • alternatives to nitrocellulose can be used, such as polyvinylidene di-fluoride (PVDF) membrane
• Antibodies: used for detection of a specific protein
  • primary antibody: specific to target protein
  • secondary antibody: used to detect the primary antibody; conjugated to a signal marker (enzyme or fluorochrome)
  • reduction of non-specific antibody binding to the nitrocellulose can be achieved using a Blocking Buffer containing non-fat dry milk and a non-ionic detergent to block non-specific sites in the protein
  • Blocking buffers with serum in lieu of non-fat milk can be used
• Chemiluminescence detection system: used to process the enzyme linked to the secondary antibody
  • most sensitive detection method
  • the most common system uses a secondary antibody is coupled to the enzyme horseradish peroxidase/HRP and an Enhanced Chemiluminescent (ECL) Reagent
  • the ECL reagent is comprised of a stable peroxide and an enhanced luminol reagent
• Imaging system: used to detect and quantify chemiluminescence or fluorescence
  • chemiluminescent signal detected either by X-ray film or a charged-coupled device (CCD) camera
  • fluorescence detected by digital camera

Figure \(\PageIndex{1}\): Indirect vs. Direct Western Blotting. In the direct method of Western blotting (left image), a "labeled" primary antibody, coupled to a marker, such as an enzyme, is used to detect a specific protein. Development of a substrate by the enzyme produces a detectable signal. In the indirect method (right image), an uncoupled primary antibody detects the protein and a labeled secondary antibody is used to detect the primary antibody. As with the direct method, the marker on the secondary antibody produces a detectable signal. The use of a secondary antibody allows for signal amplification as more than one secondary antibody can bind a primary antibody. (Indirect vs. Direct Western Blotting by Patricia Zuk; figure created in BioRender. Zuk, P. (2025))

The protocols given below outline the transfer of proteins under wet conditions using a nitrocellulose membrane, followed by protein detection using the indirect blotting method (Figure \(\PageIndex{2}\)).

Lab Protocol: Wet Transfer of Proteins to Nitrocellulose

1. Separate proteins using SDS-PAGE and carefully open the gel cassette.
2. Remove the stacking gel by scraping it away.
3. Place a piece of Whatman filter/blotting paper on top of the separating gel.
4. Invert the gel plate and carefully lift it from the gel, leaving the separating gel adhered to the filter paper.
5. Trim off the excess blotting paper.
6. Cut another piece of filter paper and a piece of nitrocellulose membrane to the same size.
7. Prepare the transfer cassette:
   1. Fill a glass container with enough Transfer Buffer ensure coverage of the nitrocellulose membrane.
   2. Open the plastic transfer cassette and place in the container.
   3. Add a transfer sponge to one half of the transfer cassette.
   4. Place the gel on the sponge with the filter paper side down.
   5. Wet the piece of nitrocellulose in Transfer Buffer. Carefully place on top of gel, ensuring no air bubbles form between the gel and the membrane.
   6. Add the second piece of filter paper and the second transfer sponge.
   7. Close the transfer cassette.
8. Fill the transfer tank with Transfer Buffer.
9. Carefully insert the Transfer cassette into the transfer tank with the hinge facing up. Ensure that the acrylamide gel faces the black/negative electrode and the nitrocellulose membrane faces the red/positive electrode.
10. Transfer at 100V for 90-120 minutes. If done at 4°C, the transfer can be left in the transfer tank overnight.

Lab Protocol: Membrane Probing

1. Following transfer, remove the transfer cassette and disassemble it.
2. Using forceps, remove the nitrocellulose membrane and place it into a plastic staining container.
   1. Optional: To confirm transfer, stain the nitrocellulose membrane with a Ponceau S solution for 5 minutes with gentle shaking.
   2. Pour off the excess stain.
   3. Wash the membrane 3 times with Wash Buffer for 5 minutes each.
3. Block non-specific sites (and remove the Ponceau S stain) by incubating the membrane in Blocking Buffer for 1 hour at room temperature or overnight at 4°C with gentle agitation.
4. Dilute the primary antibody in Blocking Buffer to the desired concentration. Incubate the membrane in primary antibody for 1-2 hours at room temperature or overnight at 4°C with gentle shaking.
5. Wash the membrane 3 times with Wash Buffer for 5-10 minutes each.
6. Dilute the secondary antibody in Blocking Buffer to the desired concentration. Incubate the membrane in secondary antibody for 1-2 hours at room temperature with gentle shaking.
7. Wash the membrane 3 times with Wash Buffer for 5-10 minutes each.

Lab Protocol: Protein Detection

1. Decant the wash buffer.
2. Add the chemiluminescent substrate to the membrane and incubate for the time recommended by the substrate manufacturer.
3. Place the membrane in an imaging system or expose to X-ray film to visualize protein bands.
4. Compare the visualized protein band to the protein ladder.

Safety Considerations

• Handle methanol, chemiluminescent substrates, and any hazardous reagents with appropriate PPE.
• Dispose of waste according to institutional guidelines.

Figure \(\PageIndex{2}\): The Western Blotting workflow. Protein Transfer (top box): Proteins are separated by size through acrylamide electrophoresis (SDS PAGE). The acrylamide gel is transferred to filter paper for increased support and then assembled into a transfer cassette, comprised of two transfer sponges surrounding filter paper, the gel, and a membrane made of nitrocellulose (or PVDF). The proteins are electrically transferred from the acrylamide gel onto the nitrocellulose. Immunoblotting (bottom box): The transfer cassette is disassembled and non-specific sites on the nitrocellulose blocked for several hours (usually with buffer containing non-fat milk or animal serum). The nitrocellulose is incubated with a buffer containing the desired primary antibody, followed by several short wash steps. The membrane is incubated with buffer containing the appropriate secondary antibody and then washed again. The secondary antibody is conjugated to a marker that can be detected. The most common marker is an enzyme like horseradish peroxidase (HRP). Protein bands in the nitrocellulose membrane bound to the primary-secondary antibody complex are visualized by developing the conjugated marker and producing a detectable signal. For HRP, the membrane is incubated with a luminol/peroxide solution that results in a light signal that can be visualized using X-ray film or a camera-based detection system. (Western Blotting by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2025))

Western Blot Reagents

Western Transfer buffer

• combine the following in 3800 mL distilled water:
  • 115.31 g Glycine
  • 24.23 g Tris base
  • 800 mL methanol
• add distilled water to 4 L
• final concentration:
  • 384 mM Glycine
  • 50 mM Tris
  • 20% methanol

Western Blocking buffer

• combine the following in 400 mL distilled water:
  • 25.0 g non-fat dry milk or 15.0 g bovine serum albumin (BSA)
  • 50 mL 10X Phospho-buffered Saline (PBS)
  • 500 uL Tween-20
• add water to 500 mL
• final concentration:
  • 5% non-fat milk or 3% BSA
  • 1X PBS
  • 0.1% Tween-20

Western Wash Buffer

• combine the following in 800 mL distilled water:
  • 100 mL 10X Tris-buffered Saline (TBS)
  • 1.0 mL Tween-20
• add distilled water to 1 L
• final concentration:
  • 1X TBS
  • 0.1% Tween-20

10X Phospho-buffered Saline (PBS)

• combine the following in 900 mL distilled water:
  • 80.0 g NaCl
  • 2.0 g KCl
  • 17.8 g Na₂HPO₄
  • 2.4 g KH₂PO₄
• add distilled water to 1 L
• 10X final concentration:
  • 1.37 M NaCl
  • 27 mM KCl
  • 100 mM Na₂HPO₄
  • 18 mM KH₂PO₄

10X Tris-buffered Saline (TBS)

• combine the following in 900 mL distilled water:
  • 24.0 g Tris
  • 88.0 g NaCl
  • adjust the pH to 7.6 with 10N HCl
  • add distilled water to 1 L
• 10X final concentration:
  • 200 mM Tris-Cl
  • 1.5 M NaCl

Ponceau S Solution

• dissolve 0.5 g of Ponceau S in 1.0 mL glacial acetic acid
• bring to 100 mL with distilled water
• final concentration:
  • 0.5% (w/v) Ponceau S
  • 1.0% (v/v) glacial acetic acid