title: A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.
abstract: The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term "western blot" suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many "optimized" western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.
authors.affiliation: Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Eaton
authors.affiliation: Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
authors.forename: Maica Llavero
authors.initials: ML
authors.lastname: Hurtado
authors.affiliation: Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
authors.forename: Karla J
authors.initials: KJ
authors.lastname: Oldknow
authors.affiliation: Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
authors.forename: Laura C
authors.initials: LC
authors.lastname: Graham
authors.affiliation: Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Marchant
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh; Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Gillingwater
authors.affiliation: Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh.
authors.forename: Colin
authors.initials: C
authors.lastname: Farquharson
authors.affiliation: Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh; Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh; t.m.wishart@ed.ac.uk.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Wishart
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D001923:Brain Chemistry; D004396:Coloring Agents; D005453:Fluorescence; D006736:Horses; D051379:Mice; D009124:Muscle Proteins / Q000032:analysis; D018482:Muscle, Skeletal / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012756:Sheep
chemical_list: D004396:Coloring Agents; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins