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Western Blot Principles and Applications

In this western blot principles and applications post we have briefly explained about western blot analysis, principle, components, western blot analysis procedure, applications, advantages and limitations.

Western Blot Analysis

Western blot analysis is an analytical technique for detecting proteins. The method uses gel electrophoresis to separate proteins based on their molecular weight (MW) and electric charge. The proteins are then transferred to a synthetic membrane and exposed to the desired antibodies (primary antibodies). The primary antibody is usually paired with a second antibody that is connected to a developer that recognizes the protein of interest.

Ulrich Laemmli invented the western blot analysis in 1970, based on the notion of molecular separation by an electrical gradient on polyacrylamide gels. Towbin (1979) described the transfer of proteins from polyacrylamide-urea gels to nitrocellulose sheets at the end of the decade, and it was Burnette two years later that coined the term western blot analysis for the approach utilizing polyacrylamide gels and sodium dodecyl sulphate (SDS-PAGE).

In the biomedical field, the western blot analysis has become an important tool for detecting viral, autoimmune, rheumatic, and oncologic illnesses.

Principle

The interaction between the proteins and the probes used to identify the proteins is the basis of western blot analysis. Gel electrophoresis is used to separate the proteins used in western blot analysis so that they can be placed on a gel matrix.

The proteins are subsequently immobilized by being transferred to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Blotting is the process of transferring protein. A reporter-labeled primary antibody directed against the protein or a reporter-labeled secondary antibody directed at the primary antibody can be used to detect the protein on the membrane.

The probe on the antibody can be an enzyme that produces a color reaction or a luminescent signal that provides a fluorescence signal in the presence of a specific substrate at the antigen-antibody binding site. The probe’s signal or color necessitates a detection system that is appropriate for the signal or intensity produced.

Requirements

Gel Electrophoresis

  1. SDS-PAGE Gel
  2. Basic Power Supply
  3. Sample buffer
  4. Sample reducing buffer
  5. Protein ladder

Protein Transfer

  1. Transfer Unit
  2. 0.45 µm nitrocellulose filter paper
  3. SDS-PAGE transfer buffer
  4. Methanol
  5. Gel knife
  6. Basic power supply

Western Blotting

  • 10x Tris-buffered saline with 1% Tween 20.
  • Shaker
  • Skim milk
  • Plastic pouches or container
  • Primary antibody
  • Secondary antibody
  • Chemiluminescent substrate
  • Gel documentation system.

Western Blot Steps

Blotting

Western Blot Principles and Applications

Tissue preparation

Whole tissue or cell culture samples are both suitable. Solid tissues are often mechanically broken down using a homogenizer or sonication. To stimulate cell lysis and solubilize proteins, a variety of detergents, salts, and buffers can be used. Protease and phosphatase inhibitors are frequently used to keep the sample from being digested by its own enzymes. To minimize protein denaturation, tissue preparation is frequently done at low temperatures.

Gel electrophoresis

SDS-PAGE (SDS polyacrylamide gel electrophoresis) keeps polypeptides denatured after treatment with strong reducing chemicals to destroy secondary and tertiary structure, allowing proteins to be separated by their molecular weight. The negatively charged SDS coats the sampled proteins, which then migrate to the positively charged electrode through the acrylamide mesh of the gel.

Smaller proteins travel quicker through this mesh, separating the proteins based on their size. The resolution of the gel is determined by the concentration of acrylamide; the higher the concentration of acrylamide, the better the resolution of lower molecular weight proteins.

Steps

Assemble the rack for gel solidification after creating the 10% stacking gel solution. Carefully pour stacking gel solution until it reaches the same level as the green bar holding the glass plates. Toss in some H2O or Ethanol on top. Allow for 15–30 minutes for the gel to solidify.

After removing the water, overlay the stacking gel with the separating gel. Make sure there are no air bubbles before inserting the comb. Wait for the gel to solidify. Fill the electrophorator with the running buffer. Connect a power supply to the electrophorator and place the gel inside. Make sure the buffer completely covers the gel before carefully removing the comb.

Fill each well with a marker (6 µL) and samples (15 µL). For separating gels, use a low voltage (60 V); for stacking gels, use a higher voltage (140 V). Run the gel for about an hour, or until the dye front flows off the gel’s bottom.

Electro transfer

Similar to Southern blot DNA transfer, the proteins are transported from within the gel onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane to make them accessible to antibody detection. Electro blotting is a method for transferring proteins that uses an electric current to draw proteins from the gel into a PVDF or nitrocellulose membrane.

The proteins travel from the gel to the membrane while preserving their structure. The proteins are exposed on a thin surface layer for detection as a result of this “blotting” process.

Steps

Cut 6 filter sheets and one polyvinylidene fluoride (PDVF) membrane to the same dimensions as the gel measurement. Using transfer buffer, wet the sponge and filter paper, and methanol, wet the PDVF membrane. Remove the gel from the glass plates by separating them and create a transfer sandwich as follows: Sponge, 3 Filter Papers, Gel PVDF, 3 Filter Papers. Transfer the sandwich to the transfer apparatus, which should be kept on ice to keep the temperature at 4°C.

Fill the device with transfer buffer, making sure the sandwich is completely coated. Place electrodes on top of the sandwich, with the PVDF membrane sandwiched between the gel and a positive electrode. Transfer for 90 minutes. (The running time should be proportional to the gel thickness, therefore for 0.75 mm gels, the time could be reduced to 45 minutes.).

Blocking

Because the membrane was chosen for its propensity to bind protein, and both antibodies and the target are proteins, precautions must be made to avoid membrane interactions with the antibody used to detect the target protein.

Non-specific binding is blocked by immersing the membrane in a dilute solution of protein, such as Bovine serum albumin (BSA) or non-fat dry milk (both of which are inexpensive), together with a little amount of detergent, such as Tween 20.

In all sites where the target proteins have not attached, the protein in the dilute solution adheres to the membrane. As a result, when the antibody is introduced, there is no place on the membrane for it to adhere to anything other than the precise target protein’s binding sites. This decreases “noise” in the western blot analysis final product, resulting in clearer results and fewer false positives.

Steps

In western blot analysis, blocking is a crucial step. Antibodies are proteins; hence they will most likely adhere to the nitrocellulose paper. So, before adding the primary antibody, casein or Bovine serum albumin is used to non-specifically saturate or mask the membrane (BSA). For 1 hour, block the membrane with 5% skim milk in TBST.

Antibody incubation

During the detection procedure, a modified antibody attached to a reporter enzyme “probes” the membrane for the protein of interest, which when exposed to a suitable substrate causes a colorimetric reaction and creates a color. This has typically been a two-step process for a variety of reasons, while there are now one-step detection approaches available for some applications.

Steps

In a shaker, mix the primary antibody with 5% bovine serum albumin (BSA) and incubate overnight at 4°C. For 5 minutes, soak the membrane in TBST. Rep this process three times. In TBST, mix the secondary antibody with 5% skim milk and incubate for 1 hour. For 5 minutes, soak the membrane in TBST. Rep this process three times.

Visualization

The western blot analysis is now ready for detection of the probes that are tagged and bound to the protein of interest after the unbound probes have been washed away.

a. Colorimetry

The western blot is incubated with a substrate that reacts with the reporter enzyme (such as peroxidase) attached to the secondary antibody in the colorimetric detection method. The soluble dye is converted to an insoluble version of a different color, which precipitates near to the enzyme and colors the membrane. The blot is then stopped from developing by washing away the soluble dye. Protein concentrations are measured using densitometry (the intensity of the stain) or spectrophotometry.

b. Chemiluminescence

Chemiluminescent detection relies on incubating the western blot with a substrate that will glow when exposed to the secondary antibody’s reporter. Photographic film, and more recently, CCD cameras, detect the light and capture a digital image of the western blot. Densitometry is used to analyses the image, determining the relative amount of protein staining and quantifying the results in terms of optical density. If proper standards are utilized, newer software allows for additional data analysis, such as molecular weight analysis.

c. Radioactive sources

Instead of requiring enzyme substrates, radioactive labels allow the insertion of medical X-ray film directly against the western blot, which develops when it is exposed to the label and produces dark patches that correspond to the protein bands of interest. Radioactive detection technologies are becoming less important because to their high cost, significant health and safety dangers, and the availability of ECL as a viable substitute.

d. Fluorescence

The fluorescently labelled probe is excited by light, and the excitation’s emission is detected by a photosensor, such as a CCD camera with appropriate emission filters, which captures a digital image of the western blot and allows for further data analysis, such as molecular weight analysis and quantitative western blot analysis. For blotting analysis, fluorescence is regarded one of the most sensitive detection modalities.

Result Interpretation

The result of western blot analysis depends on the type of probes used during the process. If an enzyme-conjugated secondary antibody is used, the reaction between the substrate and the enzyme produces a color.

The soluble dye is converted into an insoluble form, resulting in a different color on the membrane. In order to stop the development of a blot, the dye is removed by washing the membrane. The protein levels can then be evaluated by spectrophotometry.

Applications

1. Different biomarkers, such as growth factors, cytokines, and hormones, are also analysed using western blot analysis.

2. In gene expression studies, the technique has been used to quantify proteins and other gene products.

3. Western blot analysis is a highly sensitive method for detecting a specific protein, even in little quantities.

4. Western blot analysis has been used to diagnose a variety of disorders in the clinic. A western blot is used to detect anti-HIV antibodies in the serum as a confirmatory test for HIV.

Limitations

1. Due to insufficient protein transfer, no bands or erroneous bands may be identified in some circumstances of western blot analysis.

2. Because the estimation is not always precise, western blot analysis can only be used as a semi-quantitative test.

3. Proteins can only be analysed using western blot analysis if the primary antibodies for the proteins are available.

4. By interacting with more than one protein in the sample, some antibodies may have off-target effects.

5. With the expense of antibodies and sophisticated detection technologies, the approach is a pricey process.

Further Readings

Reference