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Western Blot: Definition, Methods, Functions And Its Recent Uses.

Western Blot Definition

Since the late 1970s, the Western blot test has been used to identify immune system proteins. Using a conventional methodology, Western blotting or immunoblotting may identify one or more particular protein antibodies in a sample. The electrophoresis separation of bigger molecules is the first step in the Western blot technique. These denatured molecules are subsequently blotted onto a membrane that has been created specifically for this purpose. Specific antibodies that have been tagged may then be used to recognise a specific antigen.

What is Western Blotting?

Separating antigens from a mixed protein sample using Western blotting is a viable procedure. Antigens are foreign chemicals that elicit a reaction from the immune system. The sensitivity and specificity of Western blot findings are both high, which indicates that false-negative and false-positive results are rare. Simply said, high sensitivity yields correct findings from samples containing illness evidence, whereas high specificity detects high rates in people who do not have the condition.

Even so, a Western blot result isn’t completely reliable. Antibodies may recognise a variety of antigens. For example, if someone is being tested for HIV and has a lung infection at the same time, antibodies may pick up on the respiratory illness and fail to indicate the presence of HIV. To improve test accuracy, precision is required throughout each Western blot phase.

This test looks for particular proteins in a sample of mixed tissue, blood, urine, or saliva. It’s used to identify sickness, pharmacological side effects, and doping in sports. It also plays an essential part in scientific and clinical research.

The Western blot test is used in molecular biology and is named after its predecessor, the Southern blot test, which identifies DNA sequences in DNA fragments. The Northern blot, which locates RNA molecules, is another test with a similar name.

Western blot tests follow the same stages as ELISA testing, but with different products and methodologies. These will be covered in more detail later.

Western Blot Steps

There are eight stages to performing a Western blot, all of which must be done correctly:

  • Preparation of the Western blot buffer
  • Preparation of samples
  • Gel electrophoresis is used to separate protein mixtures.
  • Separated proteins are transferred to a membrane (blotting).
  • Membrane immobilisation
  • Incubation of antibodies
  • The target protein’s detection
  • Analyzing data

Preparing the Buffer

Various buffers are required for Western blot testing to prevent changes in sample pH throughout the testing procedure. Stains and destaining solutions, such as Coomassie Brilliant Blue and Pinceau S, must be combined and diluted according to conventional formulae.

During the cell lysis (breakdown), protein separation, protein transfer, and blocking stages, buffers are used.

Sample Preparation

You’ll need a tissue sample to perform Western blot analysis. This sample will also need to be prepared. Mechanical techniques are used to break sample cells at low temperatures. Proteins do not degrade at low temperatures. Disrupting cells may be done in a variety of ways, including:

  • Homogenization is the process of combining particles to create an emulsion or solution.
  • Sonication is the process of agitating and mixing particles by employing sound energy (ultrasonic frequencies) 
  • Homogenization at high pressure is referred to as high-pressure disruption.

A buffer is then used to further break down the material. If the wrong lysis buffer is employed, the protein under investigation may not be properly extracted.

Protein Separation

Tissue-based samples are difficult to work with. They include a huge number of short polypeptide chains and bigger proteins, as well as a variety of kinds and shapes. Lipids and carbs may also be present.

From microtubules to mitochondria, every cell has organelles made partially or entirely of protein. Without laboratory procedures like Western blotting, detecting one protein type from every tissue sample is difficult.

Separating these macromolecules within a sample is the next Western blot stage. Preparation, which ruptures complete cells, takes this a step farther than protein separation. Gel electrophoresis is the gold standard for protein separation. In gel electrophoresis, an electrical current is used to push macromolecules across a gel slab.

A gel slab is sandwiched between two glass or paper plates in a standard electrophoresis chamber. The gel slab has spacers that keep the chamber consistent. The more samples that can be accommodated by a thicker slab, the less accurate the findings will be since heat will not disperse as effectively. Electrophoresis chambers are housed in an electrophoresis tank, which generates the required electric field.

One-dimensional and two-dimensional electrophoresis are the two forms of electrophoresis. Routine proteins are separated using a one-dimensional technique. Multiple protein samples may be compared at the same time and on the same gel using two-dimensional electrophoresis. Different coloured dyes are used to tag different target proteins. The one-dimensional form is described in this article.

The most prevalent techniques of protein separation in Western blot analysis are native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE (SDS-PAGE). A single resolving gel is used in these experiments. Separating gel is another term for resolving gel.

Other chemicals are added right before the test to polymerize (form molecular chains) the gel before it is run. Because oxygen interferes with the polymerization process, they are introduced without it. This necessitates degassing the electrophoresis chamber first. Polymerization alters the size of the holes that pass through the gel, improving the accuracy of the final Western blot result.

Another gel, called a “stacking gel,” lines up samples of proteins. Stacking gels operate like the starting line for a horse race, lining up competitors before they go through the resolving gel (much more slowly).

Because various proteins have varying molecular weights, they travel at different speeds through the resolving gel and onto a membrane. This establishes a barrier between them.

Protein or Western Blot Transfer

An electrical charge that surrounds the sample proteins causes protein transfer. Proteins are directed and pushed from gel to membrane under a regulated electrical field. The divided proteins “blot” onto the membrane as they reach it.

Wet or semi-dry Western blot transfer is possible. Diffusion, capillary action, and vacuum transfer are some of the slower processes. Semi-dry procedures are faster than wet electroblotting in an electrophoresis tank, while wet electroblotting in an electrophoresis tank yields more precise findings.

Charged proteins are drawn toward the blotting membrane, which is closest to the positive electrode of the gel electrophoresis tank. At the commencement of the protein separation phase, they are surrounded by negatively charged ions. Buffer chemicals push negatively-charged ions in front of and behind the proteins when an electric current is applied. The positive charge adjacent to the membrane attracts these negatively charged boundaries, which travel around the chamber at varying speeds. The molecular weight of individual proteins determines how fast they move (molecular sieving).

Finally, the divided proteins blot onto the membrane. These protein stacks, sandwiched between negatively charged ions, produce stained lines of varying thicknesses depending on sample volume; they emerge at varied levels depending on molecular weight. Pore size influences how many proteins pass through the gel slab; smaller holes block bigger proteins.

Electrophoresis chambers with pre-marked proteins with exact molecular weights are available for purchase. These are known as calibrating gels, and they’re used to figure out the molecular weights of lesser-known proteins.

Membrane Blocking

For proper Western blot findings, blocking buffers are required. Antibodies are prevented from attaching to the blotting membrane by them.

The membrane is saturated with proteins after protein blotting, yet there are still vacant spaces. Because this membrane was designed to collect and keep proteins, and because antibodies are proteins, the membrane might have an impact on the end outcome.

A Western blot result without membrane blocking might seem like background noise on a video recording, obscuring the picture. This noise is a cause of false-positive findings in Western blot blocking buffers. Antibody probing is another word for membrane blocking.

Blocking buffers fill up the membrane’s vacant gaps. Skim milk powder, surprisingly, is a frequent buffer. Blood serum and the non-protein polyvinylpyrrolidone are used to make other blocking buffers (PVP).

Antibody Incubation

Based on direct and indirect protein detection techniques, there are two types of antibody incubation. A single (primary) antibody is used to detect a single protein in a direct Western blot. The tag is a dye or enzyme that makes the findings visible. This tagged antibody attaches to the target protein.

The indirect Western blot approach employs two antibodies: a primary antibody that binds to the target protein and a tagged secondary antibody that detects and designates the primary antibody’s location on the blot, allowing it to be seen.

Antibodies (immunoglobulins) are produced in large amounts in genetically engineered animals. Monoclonal antibodies are generated entirely by humans. They exclusively detect a single sort of antigen epitope, which is the portion of an antigen that immunoglobulins bind to. In many circumstances, monoclonal antibodies are more costly than polyclonal antibodies, but they produce more accurate and reliable findings.

Polyclonal antibodies groups that recognise several epitopes are known as immunoglobulins (Ig). On the other hand, each antibody in this category functions like a monoclonal Ig, detecting just one epitope on a single antigen. When polyclonal antibodies are employed in Western blots, the test is less expensive, but there is a greater danger of cross-reactivity, which is the identification of antigens that are similar but not identical.

The time it takes for a primary antibody to bind to a target protein is determined by its binding affinity. The average time is two hours at room temperature (20°C or 68°F) or overnight at 4°C (39.2°F). Longer incubation durations result in greater signal noise.

The membrane must be washed several times in the appropriate wash solution before adding the secondary antibody for indirect incubation. The incubation periods for secondary antibodies are similar to those for primary antibodies.

Detection of the Target Protein

Detection methods for Western blot procedures vary depending on the antibody tags employed. Antibody labels may be recognised using X-ray film, light sensor cameras (charge-coupled devices), fluorescent digital imagers, and near-infrared spectroscopy tools.

All primary or secondary antibodies must be washed from the membrane before detection. This eliminates the possibility of future responses.

For proper analysis of the data, good protein visualisation is required. Human mistakes or contaminated, malfunctioning, or out-of-date solutions, materials, and equipment are the most common causes of poor protein detection.

Data Analysis

Nowadays, Western blot data processing frequently necessitates the digitization of membrane scans.

Protein band values are calculated by comparing them to a marker whose molecular weight is known.

Several key elements must be considered throughout the analysis:

  • Limits of detection: Certain proteins are more difficult to detect than others.
  • Other effects, such as bacterial contamination, might cause signals to weaken or even vanish over time.
  • When the detection limit is high, the dynamic range – the ratio of protein amount to signal intensity – is linear.
  • Signal-to-noise ratio: proteins that are related to target proteins are also found, creating background noise for any target-protein signals.

Western Blot Analysis

While the findings of Western blot analysis are typically used to identify the presence of illness, they may also be used for other medical purposes. For example, pharmaceutical research examines how medications influence different tissues. Many firms specialise in Western blot testing antibodies. However, our understanding of particular antibodies is limited, and whether the antibodies generated can detect less-studied antigens is a point of contention.

Animals are genetically engineered to lack the gene that encodes the protein under study in order to develop Western blot antibodies. When an animal is exposed to it, the immune system recognises the protein as a foreign intruder and produces antibodies to eliminate it. A knockout animal is one that has been genetically engineered. Rather than purchasing ready-made antibodies, many labs create their own knockout mice.

It’s not difficult to figure out how to interpret a Western blot. A good or negative outcome is assigned to the bands. Not all of the outcomes are obvious. A number preceded by the letter p or followed by kDa is used to denote band sizes (protein molecular weight in kilodaltons). The p stands for the protein density inside the sample that has been defined.

Western blot analysis The proteins of the human immunodeficiency virus are identified by HIV tests. It detects four proteins: glycoproteins 160 and 120, as well as the antigens p24 and p31. At least one of each glycoprotein and antigen set must be present in positive findings.

Western blot Lyme findings are 97 percent accurate for this tick-borne illness, but only in those who have had Lyme arthritis or Lyme carditis. Borrelia burgdorferi, which is transmitted via the blood, is the source of the identified bacterial proteins.

The Western blot herpes detection test developed by the University of Washington used to be the gold standard in testing, but it has subsequently been demonstrated to yield excessively high false-positive findings. Because further testing is often necessary, this is a costly screening procedure.

Western Blot vs ELISA

The enzyme-linked immunosorbent test (ELISA) is the polar opposite of the Western blot. ELISA examines the presence of antibodies by attaching them to known antigens, rather than detecting antigens via antibodies.

Because there are no viruses remaining in the body, a Western blot will not detect viral antigens if a person has been infected with a virus in the past but is no longer unwell. ELISA, on the other hand, may identify antibodies created after a previous infection.

The use of N195 protein as an antigen for early Western blot detection of an active SARS coronavirus infection is presently being investigated. In contrast, an ELISA kit for detecting the presence of two kinds of COVID-associated immunoglobulins is currently available. If IgM is found, it implies that the illness is in its early stages. IgG detection in the absence of aberrant IgM levels indicates a previous infection.

References

  • Burgess RR, Deutscher MP (Eds). (2009). Methods in Enzymology: Volume 463, Guide to Protein Purification. San Diego, Elsevier Science Publishing Co Inc.
  • Mahmood T, Yang, PC. (2012). Western Blot: Technique, Theory, and Troubleshooting. North American Journal of Medical Sciences. 4(9), 429–434. https://doi.org/10.4103/1947-2714.100998.
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