An overview of technical considerations for Western blotting applications to physiological research

Abstract

The applications of Western/immuno-blotting (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, also 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.

Sarcolipin expression in human skeletal muscle: Influence of energy balance and exercise

“…The gels were incubated overnight at 4°C. The same control extract was loaded in all gels to assure comparability between gels . Proteins were transferred to Immun‐Blot PVDF Membranes and subsequently stained with Reactive Brown 10 to control for differences in loading and transfer efficiency, and the bands captured with the ChemiDocTM Touch Imaging System and quantified with the Image Lab 5.2.1 software.…”

Western Blot Analysis of Cells Encapsulated in Self-Assembling Peptide Hydrogels

“…The detergent SDS is widely used as an effective agent for membrane disruption and the solubilization of cellular proteins (2). SDS is an anionic surfactant that acts to both coat hydrophobic regions of proteins with a negative charge and mask positive charges in proteins.…”

“…Over the last 40 years (1), western blot analysis has become a wide-spread technique used for a range of purposes, including identification and quantification of proteins, determination of proteinprotein interactions, and detection of post-translational proteins modifications, to name a few (2). The term western blot is used to describe the entire process from protein extraction, solubilization, and size separation through to membrane transfer and subsequent antibody detection (3).…”

“…The choice of buffer depends on sample type (57), protein of interest (8), and downstream application. Typical buffer components include detergents (anionic, cationic, and non-ionic), salts, reducing agents, pH-buffering compounds, and protease and phosphatase inhibitors (2). Sample-specific optimization is an intrinsic part of method development.…”

Estudio epidemiológico de hidatidosis comparando los test de ELISA IgE y Western Blot en población de cinco provincias de la Región Lima. 2015

“…En relación a las diferencias encontradas en la frecuencia de anticuerpos IgE (6,96%) e IgG (5,89%) en el presente estudio, una posible explicación es que se utilizaron antígenos en estado nativo para sensibilizar las placas de ELISA IgE, no han sufrido variación de estructura a diferencia de los antígenos utilizados para la prueba de Western Blot IgG, que son previamente tratados con el detergente SDS a altas temperaturas (95°C) que desconfigura la estructura tridimensional de las proteínas antigénicas eliminando potencialmente los epítopes conformacionales que normalmente están presentes en el estado nativo del antígeno, quedando expuestos solo los epítopes lineales (19,20) . Una concordancia del 99% entre la ocurrencia de anticuerpos IgG e IgE demuestra que la mayoría de estos individuos expuestos producen en alguna medida ambos tipos de anticuerpos contra los antígenos del parásito.…”