NDLI: Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes

Content Provider	PubMed Central
Author	Murphy, Robyn M. Lamb, Graham D.
Copyright Year	2013
Abstract	Western blotting has been used for protein analyses in a wide range of tissue samples for >30 years. Fundamental to Western blotting success are a number of important considerations, which unfortunately are often overlooked or not appreciated. Firstly, lowly expressed proteins may often be better detected by dramatically reducing the amount of sample loaded. 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). That is an order of magnitude less than often used. 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. Secondly, incorrect results can be obtained if samples are fractionated and a proportion of the protein of interest inadvertently discarded during sample preparation. Thirdly, quantitative analyses demand that a calibration curve be used. This is regardless of using a loading control, which must be proven to not change with the intervention and also be appropriately calibrated. Fourthly, antibody specificity must be proven using whole tissue analyses, and for immunofluorescence analyses it is vital that only a single protein is detected. If appropriately undertaken, Western blotting is reliable, quantitative, both in relative and absolute terms, and extremely valuable.
Related Links	http://dx.doi.org/10.1113/jphysiol.2013.263251
Ending Page	5831
Page Count	9
Starting Page	5823
File Format	PDF
ISSN	00223751
e-ISSN	14697793
Journal	The Journal of Physiology
Issue Number	Pt 23
Volume Number	591
Language	English
Publisher	Blackwell Science Inc
Publisher Date	2013-12-01
Access Restriction	Open
Rights Holder	Blackwell Science Inc
Subject Keyword	Physiology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Physiology Sports Science

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