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ANALYSIS OF WESTERN BLOT PROCEDURES

Proteins play many vital roles in our bodies. Essentially proteins or polypeptides are complex compounds of amino acids that are needed by our bodies to regulate, function and construct organs and tissues in the body.

Proteins are integral to organisms, helping almost every cell process and are also critical to metabolism. For these reasons, detecting the presence or absence of protein, their size or molecular weight is vital. Protein analysis is crucial in

investigating a disease,

detecting the presence of allergenic protein in food samples and

evaluating whether genetic manipulation experiments were successful or not

There are different types of proteins in terms of their size and the arrangement of amino acids, having diverse molecular structures, nutritional characteristics, and chemical properties. Given the diverse nature of this vital biological element, there are three traditional techniques that researchers and scientists use for protein analysis:

protein separation,

western blotting, and

protein identification.

In this blog, our team at HHC will help you explore the western blot technique to understand the procedure and its uses for protein analysis

WHAT IS WESTERN BLOTTING?

Developed by Harry Towbin and his colleagues in 1979, the western blot procedure is a typical cell and molecular biology procedure widely used in the analysis of proteins. Often known as protein immunoblotting, the method allows researchers to determine the presence, size and quantity of particular proteins in a given sample. Three elements comprise the western blotting method:

1) separation by size, 2) transfer to a solid support or membrane, and 3) marking target protein using a proper primary and secondary antibody to visualise.

Western blotting is very useful for identifying individual proteins from a complicated mixture of proteins isolated from cells that may have similar characteristics or sizes.

THE WESTERN BLOT PRINCIPLE

The Western blotting technique is based on the use of polypropylene gel electrophoresis and antibodies. In this technique, researchers can separate and identify proteins based on their molecular weight and type from a gel-like sample using electrophoresis, which acts as a molecular sieve. Next, the separated proteins are transferred to a membrane that produces a band for each protein. The membrane is then incubated with labelled antibodies specific to the protein of interest to create a coloured band. These antibodies can be identified, and the size and abundance of the bound proteins can be analysed in relation to established standards or controls.

HOW IS THE WESTERN BLOT PROCEDURE PERFORMED?

The western blotting technique is performed in 7 steps. They are

1) Sample preparation

2) Gel electrophoresis

3) Proteins transfer

4) Blocking

5) Primary Antibody incubation

6) Secondary Antibody incubation

7) Protein detection & analysis

1) Sample preparation

The first step in the western blotting technique involves the extraction of proteins from different samples such as cells or tissues. The extracted samples need to be broken down by a homogeniser or sonication. Phosphatase inhibitors and protease are used to avoid the digestion of the sample at cold temperatures. Once the protein is extracted, the quantity of proteins needs to be determined.

2) Gel electrophoresis

This step involves the separation of the proteins based on their charge and molecular weight. This is done using polypropylene gel electrophoresis like sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) or native PAGE. Usually, separating gels come in 5%, 8%, 10%, 12% or 15%. The size of the prey protein is taken into consideration when choosing the right percentage of separating gel. The relation is inverse. Higher percentage gels are suitable for proteins that weigh less.

3) Proteins transfer or blotting

Once the proteins are separated using gel electrophoresis, they are transferred to a solid support membrane such as nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane to facilitate antibody probing. Protein transfers can be performed using various techniques: diffusion transfer, capillary transfer, and vacuum blotting. But the most used is the electroblotting technique, wherein an electric current is applied to the protein gel sandwiched between the membrane and the filter papers. An electric field is aimed perpendicular to the gel’s surface that extracts the protein from the gel and transfers them onto the membrane in a tight manner.

Protein gel transfer through electroblotting can be performed in different ways, such as wet, semi-dry and dry transfer. Wet or semi-dry transfer strategies usually provide reliable results as the buffering is more flexible. However, these methods are time-consuming. On the other hand, the dry transfer method is efficient and quick but offers less buffering flexibility.

4) Blocking

The next and crucial step in the western blot procedure is blocking. Because blotting membranes have a high affinity for proteins, blocking any residue binding sites after the transfer is critical to avoid further non-specific binding of the assay detection antibodies. This is accomplished by exposing the membrane to a liquid that contains protein, such as milk or serum. BSA and non-fat dry milk are the most used typical blockers.

When submerging the membrane in the diluted protein solution, the proteins in the solution bind to all the areas of the membrane that are not bound by the prey protein. The process reduces the “noise” in the western blot’s result to provide more precise findings.

After blocking, antibody probing is performed. There are two strategies for this:

direct detection using a single or primary antibody and

indirect detection using a secondary antibody

For the western blot technique, the indirect method is used most often.

5) Primary Antibody incubation

At this stage, the primary antibody is used to probe the membrane and binds to the prey protein. Depending on the antigen to be determined, the primary antibody is selected.

In the direct detection method, the membrane is then incubated with the secondary antibody to bind to the primary antibody and is detectable.

6) Secondary Antibody incubation

When using indirect assay detection, it is vital to wash the membrane using an antibody-buffer solution to reduce background and remove excess or unbound antibodies before the secondary antibody is used to incubate the membrane. After the wash, the specific enzyme-conjugated secondary antibody incubates the membrane. When the second antibody incubation is performed, the labelled secondary antibody binds to the primary antibody that has interacted with the prey protein and is detectable. The selection of the suitable secondary antibody is based on the variety or the species of the primary antibody.

7) Protein detection & analysis

At this final stage, protein detection is achieved when the conjugated enzyme interacts with a substrate introduced to the membrane, causing a chemical change in the form of a coloured substance allowing the researcher to mark the location and the densitometry of the prey protein. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most used enzymes. Due to its stability, suitability for most conjugations, and affordability, HRP is frequently preferred.

Protein detection and the subsequent visualisation can be performed using chemiluminescent, colourimetric, radioactive and fluorescent detection techniques, the most common being chemiluminescent detection.

When using the chemiluminescent detection method, it is essential to note that the duration of the signal or the chemical change lasts only until the substrate reacts with the conjugated enzyme (usually between 1 and 24 hours), making it a very sensitive technique. Creating a permanent record of the signal is possible by using an X-ray film or using digital imaging.

WHAT ARE THE USES OF THE WESTERN BLOTTING TECHNIQUE?

In brief, the western blotting procedure determines the presence or absence, size, and abundance of target proteins in a sample which is beneficial for various scientific reasons across many fields of study. In this regard, the technique proves beneficial to evaluate the following:

Protein DNA interactions

Protein-protein interactions

Post-translational modifications (PTMs)

Protein isoform detection

Antibody characterisation

Epitope mapping

Subcellular protein localisation

Among all the applications of the Western blotting method, the most common are as below.

Diagnosis of infectious diseases

Although time-consuming when compared to other alternatives like the PCR technique, Western Blot proves highly useful in testing and confirming cases of HIV (human immunodeficiency virus), BSE (bovine spongiform encephalopathy), Lyme disease and aspergillosis.

Biomarking non-infectious diseases

As Western Blot is an antibody-based method, the technique often supports and produces reliable results for detecting non-infectious diseases using HTS (High throughput screening). For example, when determining cancers, incongruous isoforms of proteins can become potential markers of the pathology of the disease. Autoantibodies may also indicate an autoimmune disease.

Identifying the differences in protein levels between groups or over time

Western Blot is very efficient in determining changes in protein levels or even different cycle phases for samples studied in a temporal experiment. Additionally, contrasting samples from various disease or treatment groups can draw attention to protein-level changes that could point to a disease’s underlying aetiology or impact a treatment’s effectiveness.

Determine if a protein is expressed

When performing experimental genomic manipulations, Western Blot can help compare the changes made at the genomic level with those that occur at protein levels. (for example, is a novel protein expressed in the expected tissue or location? Or is its expression truncated or lost compared to a wild-type strain?).

Tagged protein detection

Tags can be engineered and assigned to proteins during genomic manipulations facilitating the identification of proteins attached to them from samples of a system under study or of a species. This method enables researchers to localise the protein and understand its course. The process can prove particularly useful for checking recombinant proteins produced through experiments.

Compared to methods like PCR (polymerase chain reaction), western blotting is not usually considered a quantitative procedure. However, the direct identification of proteins it delivers is efficient and has proven significantly advantageous for confirmatory testing for disease diagnosis.

The key to achieving reliable results from a successful blot procedure is the selection of the correct antibody and identifying the optimal concentration. The use of the right equipment is equally essential to get optimised results.

HHC provides in the ZeptoMetrix WESTERN BLOT for in vitro detection of antibodies to SIV (Simian Immunodeficiency Virus, the lentivirus most closely related to Human HIV) in serum or plasma. It is available in 10 or 30 strip kit formats. Additionally, we offer a bench top, western blot processor: the AUTOBLOT 3000, which controls incubation times, dispensing volumes and washing programs. The AUTOBLOT 3000 can programme ten user-defined protocols.

If you want to add the most updated lab equipment to enhance the protein detection services of your lab, don’t hesitate to get in touch with Helvetica Health Care Website now!

Originally Posted On HHC

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