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The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish and quantify the different proteins in a complicated protein combination. Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognizes and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognizes and binds to the primary antibody. The secondary antibody is visualized through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein. Applications

The western blot is extensively used in biochemistry for the qualitative detection of single proteins and protein-modifications (such as post-translational modifications). At least 8–9% of all protein-related publications are estimated to apply western blots. It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins .It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins. A semi-quantitative estimation of a protein can be derived from the size and color intensity of a protein band on the blot membrane. In addition, applying a dilution series of a purified protein of known concentrations can be used to allow a more precise estimate of protein concentration. The western blot is routinely used for verification of protein production after cloning. It is also used in medical diagnostics, e.g., in the HIV test or BSE-Test.

Identification of protein localization across cells
For medication development, the identification of therapeutic targets, and biological research, it is essential to comprehend where proteins are located within a cell. The subcellular locations of proteins inside the cell and their functions are closely related. The relationship between protein function and localization suggests that when proteins move, their functions may change or acquire new characteristics. A protein's subcellular placement can be determined using a variety of methods. Numerous efficient and reliable computational tools and strategies have been created and used to identify protein subcellular localisation. With the aid of subcellular fractionation methods, WB continues to be an important fundamental method for the investigation and comprehension of protein localisation.

Epitope mapping
Due to their various epitopes, antibodies have gained interest in both basic and clinical research. The foundation of antibody characterization and validation is epitope mapping. The procedure of identifying an antibody's binding sites (epitopes) on the target protein is referred to as "epitope mapping." Finding the binding epitope of an antibody is essential for the discovery and creation of novel vaccines, diagnostics, and therapeutics. As a result, various methods for mapping antibody epitopes have been created. At this point, western blotting's specificity is the main feature that sets it apart from other epitope mapping techniques. There are several application of western blot for epitope mapping on human skin samples, hemorrhagic disease virus.

Sample Preparation
As a significant step in conducting a western blot, sample preparation has to be done effectively since the interpretation of this essay is influenced by the protein preparation, which is composed of protein extraction and purification processes. To achieve efficient protein extraction, a proper homogenization method needs to be chosen due to the fact that it is responsible for bursting the cell membrane and releasing the intracellular components. Besides that, the ideal lysis buffer is needed to acquire substantial amounts of target protein content because the buffer is leading the process of protein solubilization and preventing protein degradation. After completing the sample preparation, the protein content is ready to be separated by the utilization of gel electrophoresis.

Secondary probing[edit]
One major difference between nitrocellulose and PVDF membranes relates to the ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes. While there are well-established protocols available for stripping nitrocellulose membranes, the sturdier PVDF allows for easier stripping, and for more reuse before background noise limits experiments. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and more resistant to damage during use.

Minimum requirement specification for Western Blot
In order to ensure that the results of Western blots are reproducible, it is important to report the various parameters mentioned above, including specimen preparation, the concentration of protein used for loading, the percentage of gel and running condition, various transfer methods, attempting to block conditions, the concentration of antibodies, and identification and quantitative determination methods. Many of the articles that have been published don't cover all of these variables. Hence, it is crucial to describe different experimental circumstances or parameters in order to increase the repeatability and precision of WB. To increase WB repeatability, a minimum reporting criteria is thus required.

Detection problems
There may be a weak or absent signal in the band for a number of reasons related to the amount of antibody and antigen used. This problem might be resolved by using the ideal antigen and antibody concentrations and dilutions specified in the supplier's data sheet. Increasing the exposition period in the detection system's software can address weak bands caused by lower sample and antibody concentrations.

Multiple band problems
When the protein is broken down by proteases, several bands other than predicted bands of low molecular weight might appear. The development of numerous bands can be prevented by properly preparing protein samples with enough protease inhibitors. Multiple bands might show up in the high molecular weight region because some proteins form dimers, trimers, and multimers; this issue might be solved by heating the sample for longer periods of time. Proteins with post-translational modifications (PTMs) or numerous isoforms cause several bands to appear at various molecular weight areas. PTMs can be removed from a specimen using specific chemicals, which also remove extra bands.

High background
Strong antibody concentrations, inadequate blocking, inadequate washing, and excessive exposure time during imaging can result in a high background in the blots. A high background in the blots could be avoided by fixing these issues.

Irregular and uneven bands
It has been claimed that a variety of odd and unequal bands, including black dots, white spots or bands, and curving bands, have occurred. The block dots are removed from the blots by effective blocking. White patches develop as a result of bubbles between the membrane and gel. White bands appear in the blots when main and secondary antibodies are present in significant concentrations. Because of the high voltage used during the gel run and the rapid protein migration, smiley bands appear in the blots. The strange bands in the blot are resolved by resolving these problems.

Improvements for Western Blot Related Problems
During the western blotting, there could be several problems related to different steps of this essay. Those problems could originate from a protein analysis step such as the detection of low- or post-translationally modified proteins. Additionally, they can be based on the selection of antibodies since the quality of the antibodies is playing a significant role in the detection of proteins specifically. On account of the presence of these kinds of problems, a variety of improvements are being produced in the fields of preparation of cell lysate and blotting procedures to build up reliable results. Moreover, to achieve more sensitive analysis and overcome the problems associated with western blotting, several different techniques have been developed and utilized, such as far-western blotting, diffusion blotting, single-cell resolution western blotting, and automated microfluidic western blotting.

Enhanced Western Blot
Since 1980, western blot has emerged as the most used method in molecular biology for determining the presence and quantity of a certain protein. Over the years, plenty of "advanced" and "optimized" systematical methods have been developed. These developments provide advanced and more sensitive results, with the aid of more advanced imaging technologies and modern fluorescent labeling methods.

Shift-western blot
The method with the highest adoption rate for determining DNA-binding proteins and protein-DNA interactions is the electrophoretic mobility shift test. Protein-DNA complexes are analyzed via shift-WB. It is created by transferring protein-DNA complexes, in which the DNA in the charged membrane is positioned beneath the nitrocellulose membrane while the proteins are kept in the membrane. Then, specific antibodies are used to identify the proteins, and a radioactive label is used to identify the DNA. Furthermore, the transmitted proteins and DNA can be retrieved and examined in greater detail.

Single-cell western blot
The single-cell WB (sc WB), in addition to the conventional WB, is regarded as a breakthrough in the study of protein subcellular localization and in the evaluation of single-cell protein. When measuring the levels and conditions of protein expression from one cell to the next, it is used. With the aid of single-cell WB, the western blot's selectivity and specificity were expanded to include single-cell protein analysis. The limitations of antibody accuracy and sensitivity are overcome by this technique. Furthermore, because of its versatility, it may be utilized to measure numerous target proteins concurrently from different cell lines and single cells.

Quantifiable fluorescence-based western blot
The development known as quantifiable fluorescence-based WB (QFWB) enables researchers to carry out comparative expression analysis with better sensitivity and precision than ever before. Quantifiable in QFWB refers to genuinely quantitative with increased sensitivity. This method is employed to identify the minute expression variations between various samples. With the aid of a secondary antibody that has been fluorescently tagged, QFWB produces a linear detection profile. Modern QFWB techniques enable simultaneous dual labeling and are more sensitive to identify minute variations.

Quantitative computerized western blot
Quantitative computerized western blot analyzes the reactivity of individual antibodies to specific antigens to identify immunodominant and immunorecessive determinants using two measures, such as net band intensity and total lane intensity of the WB. The creation of quick serodiagnostic tests and efficient vaccines is made possible by the identification of certain immunodominant antigens.The study looks for serological markers for the early diagnosis of cancer, viral, and autoimmune illnesses using quantitative computerized western blot.

High-throughput western blot (DigiWest)
It is a technique that combines conventional SDS-PAGE protein resolution with a bead-based microarray platform that immobilizes proteins on microspheres. This combination of protein separation, uniformity, and sensitivity allows for the quick quantification of a number of different protein targets as well as their changes. The benefit of DigiWest is that western blot is carried out using beads-based microarrays, allowing for the simultaneous detection and analysis of hundreds of distinct proteins and their changes using a wide range of varied antibodies.

Microfluidic western blot
In order to detect many proteins on a single microfluidic chip, microfluidic western blot is carried out using a number of processes, including sample enrichment, protein size, deposition of protein, and then in situ antibody probing. A photoreactive (UV light) polyacrylamide gel and a photopatternable (blue-light) surface are the foundation of this multistep procedure. Due to improvements in analytical performance, WB may now be completed in 10–60 minutes while maintaining high sensitivity detection limits (50 picomoles) and multiplexed component detection levels (femtograms). Therefore, by fusing superb specificity and the high-throughput benefits of multiplexing, WB creates a cornerstone for quick proteomics.

Multistrip western blot
A enhanced WB technique called multistrip WB is based on the simultaneous transfer of different proteins from a number of polyacrylamide gel strips to a single polyvinylidene difluoride or nitrocellulose membrane. Multistrip WB allows for the simultaneous monitoring of up to nine separate proteins from the same loading of the sample and up to a tenfold increase in data output for a single WB cycle. Systems biology, cell signaling research, and biomedical diagnostics would all benefit from using this technique.

Microchip capillary electrophoresis-based western blot
The capillary and microchip electrophoresis-based western blot was created to reduce the quantity of protein samples and the amount of time it takes to execute western blot. It contributes to the more sensitive and accurate measurement of various protein targets from any single-cell lysate carried out in a microchip. 400 nanogram of cell lysate is all that is needed to identify and quantify eleven different proteins.

Applications
In addition to the application of western blot in scientific research, it is also utilized in clinical research areas. Since it can be applied to the direct protein identification process, western blot is regarded as a powerful diagnostic tool that is frequently used in the clinic setting. WB and protein detection techniques can be used to find disease biomarkers like specific proteins or antibodies.It is thought to be a viable method for identifying particular proteins during the diagnosis of diseases like cancer, autoimmune disease, and prion disorders. The detection of several biomarkers used in the diagnosis of neurological and oncological illnesses by Western blotting is a common procedure. For instance, It is widely believed that the advent of multidrug resistance (MDR) has made effective cancer therapy extremely challenging. Therefore, early, accurate, and sensitive MDR mechanism discovery is essential, as is the search for more effective chemotherapeutic approaches for application in clinical settings. The expression of MDR-1/P-glycoprotein in the P388/ADR, P388 and HCT-15 cell lines is examined using the WB technique. WB has also identified MRP1 levels.

On the other hand, as western blot has the potential to distinguish different protein isoforms, it may be used to diagnose prion and protein isoform-related diseases, such as cancer. For instance, the WB analysis of the isoform pattern of 14-3-3 proteins in cerebral fluid can identify Creutzfeld-Jacob disease. Farmers lung disease is a pulmonary condition brought on by breathing antigenic particles, and studies have indicated that WB may be a useful option for identifying immunoreactive proteins related to farmers lung disease. Besides, western blot is also used to identify proteins in synovial fluid and serum, enabling the diagnosis of osteoarthritis and rheumatoid arthritis clinical symptoms. WB is used to assess the levels of FSTL1 protein expression in individuals with knee osteoarthritis, which serves as a potential biomarker of articular damage.