Graham Lamb is a Professor at La Trobe University in Melbourne and holds a Senior Principal Research Fellowship from the National Health and Medical Research Council of Australia. Her work is also directed at understanding calcium dependent processes in muscle and the regulation of glycogen metabolism in skeletal muscle. This approach bypasses the ambiguity in findings that typically occur when investigating heterogeneous tissue such as skeletal muscle. She has a particular focus on understanding the biochemical properties of muscle at the single cell level. Her broad research interests are in the regulation of skeletal muscle function in health and disease. Robyn Murphy is a researcher and academic based at La Trobe University in Melbourne Australia. If appropriately undertaken, Western blotting is reliable, quantitative, both in relative and absolute terms, and extremely valuable. Fourthly, antibody specificity must be proven using whole tissue analyses, and for immunofluorescence analyses it is vital that only a single protein is detected. This is regardless of using a loading control, which must be proven to not change with the intervention and also be appropriately calibrated. Thirdly, quantitative analyses demand that a calibration curve be used. Secondly, incorrect results can be obtained if samples are fractionated and a proportion of the protein of interest inadvertently discarded during sample preparation. Using heterogeneous skeletal muscle as the tissue of representation, the need to undertake Western blotting in sample sizes equivalent to single fibre segments is demonstrated. That is an order of magnitude less than often used. Single cell (fibre) Western blotting demonstrates the ability to detect proteins in small sample sizes, 5–10 μg total mass (1–3 μg total protein). Firstly, lowly expressed proteins may often be better detected by dramatically reducing the amount of sample loaded. Fundamental to Western blotting success are a number of important considerations, which unfortunately are often overlooked or not appreciated. Western blotting has been used for protein analyses in a wide range of tissue samples for >30 years.
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