Review

Authors: Rasheed Sule, Gabriela Rivera, Aldrin V Gomes...

Western blotting (immunoblotting) is a powerful and commonly used technique that is capable of detecting or semiquantifying an individual protein from complex mixtures of proteins extracted from cells or tissues. The history surrounding the origin of western blotting, the theory behind the western blotting technique, a comprehensive protocol and the uses of western blotting are presented. Lesser known and significant problems in the western blotting field and troubleshooting of common problems are highlighted and discussed. This work is a comprehensive primer and guide for new western blotting researchers and those interested in a better understanding of the technique or getting better results.

Topics: Blotting, Western; Immunoblotting; Proteins; Electrophoresis, Polyacrylamide Gel

PubMed: 36971113
DOI: 10.2144/btn-2022-0034

Review

Authors: Manish Mishra, Shuchita Tiwari, Aldrin V Gomes...

Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

Topics: Animals; Blotting, Western; Electrophoresis, Capillary; Humans; Proteins; Proteomics; Reproducibility of Results

PubMed: 28974114
DOI: 10.1080/14789450.2017.1388167

Review

Authors: Habeebunnisa Begum, Periyasamy Murugesan, Anjana Devi Tangutur...

Western blotting (WB), also known as immunoblotting, is a well-known molecular biology method that biologists often use to investigate many features of the protein, ranging from basic protein analysis to disease detection. WB is simple, unique, rapid, widely used routine tool with easy interpretation and definite results. It is being used in various fields of science, research and development, diagnostic labs and hospitals. The principle of WB is to accomplish the separation of proteins based on molecular weight and charge. This review addresses in detail the individual steps involved in the WB technique, its troubleshooting, internal loading controls, total protein staining and its diverse applications in scientific research and clinical settings, along with its future perspectives.

Topics: Biomedical Research; Blotting, Western; Proteins; Staining and Labeling

PubMed: 35775367
DOI: 10.2144/btn-2022-0003

Review

Authors: Sean C Taylor, Anton Posch

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

Topics: Blotting, Western; Densitometry; Luminescent Measurements; Proteins; Statistics as Topic

PubMed: 24738055
DOI: 10.1155/2014/361590

Review

Authors: Lakshmi Pillai-Kastoori, Amy R Schutz-Geschwender, Jeff A Harford...

Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein extraction method, electrophoresis, and transfer of proteins, immunodetection of blotted protein by antibodies, and the ultimate step of imaging and analyzing the data is nothing short of a symphony. Like with any other technology in life-sciences research, Western blotting can produce erroneous and irreproducible data. We provide a systematic approach to generate quantitative data from Western blot experiments that incorporates critical validation steps to identify and minimize sources of error and variability throughout the Western blot process.

Topics: Blotting, Western; Humans; Limit of Detection; Proteins; Reference Values; Reproducibility of Results

PubMed: 32007473
DOI: 10.1016/j.ab.2020.113608

Review

Authors: Biji T Kurien, R Hal Scofield

Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of protein patterns from gel to microporous membrane. Electrophoretic as well as non-electrophoretic transfer of proteins to membranes was first described in 1979. Protein blotting has evolved greatly since the inception of this protocol, allowing protein transfer to be accomplished in a variety of ways.

Topics: Blotting, Western; Buffers; Immobilized Proteins; Membranes, Artificial

PubMed: 26043986
DOI: 10.1007/978-1-4939-2694-7_5

Review

Authors: Gholam Hossein Meftahi, Zahra Bahari, Ali Zarei Mahmoudabadi...

Western blot (WB) or immunoblot is a workhorse method. It is commonly used by biologists for study of different aspects of protein biomolecules. In addition, it has been widely used in disease diagnosis. Despite some limitations such as long time, different applications of WB have not been limited. In the present review, we have summarized scientific and clinical applications of WB. In addition, we described some new generation of WB techniques.

Topics: Blotting, Western; Humans; Proteins; Translational Research, Biomedical

PubMed: 33847452
DOI: 10.1002/bmb.21516

Review

Authors: Yizhe Zhang, Isao Naguro, Amy E Herr...

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.

Topics: Blotting, Western; Cell Culture Techniques; Humans

PubMed: 31390130
DOI: 10.1002/anie.201906920

Review

Authors: J J Bass, D J Wilkinson, D Rankin...

The applications of Western/immunoblotting (WB) techniques have reached multiple layers of the scientific community and are now considered routine procedures in the field of physiology. This is none more so than in relation to skeletal muscle physiology (i.e., resolving the mechanisms underpinning adaptations to exercise). Indeed, the inclusion of WB data is now considered an essential aspect of many such physiological publications to provide mechanistic insight into regulatory processes. Despite this popularity, and due to the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and subsequent analysis and interpretation of the data can be variable, perhaps resulting in spurious conclusions. This may be due to poor laboratory technique and/or lack of comprehension of the critical steps involved in WB and what quality control procedures should be in place to ensure robust data generation. The present review aims to provide a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting and detection, to analysis of the data collected. We aim to provide the reader with improved expertise to critically conduct, evaluate, and troubleshoot the WB process, to produce reproducible and reliable blots.

Topics: Blotting, Western; Data Accuracy; Humans; Muscle, Skeletal; Physiology; Specimen Handling

PubMed: 27263489
DOI: 10.1111/sms.12702

Authors: Sean C Taylor, Thomas Berkelman, Geetha Yadav...

Chemiluminescent western blotting has been in common practice for over three decades, but its use as a quantitative method for measuring the relative expression of the target proteins is still debatable. This is mainly due to the various steps, techniques, reagents, and detection methods that are used to obtain the associated data. In order to have confidence in densitometric data from western blots, researchers should be able to demonstrate statistically significant fold differences in protein expression. This entails a necessary evolution of the procedures, controls, and the analysis methods. We describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods.

Topics: Animals; Blotting, Western; Densitometry; HeLa Cells; Humans; Luminescent Measurements; Proteins; Reference Standards; Reproducibility of Results

PubMed: 23709336
DOI: 10.1007/s12033-013-9672-6