title: Utility of a direct dual-mode development analysis on blotted protein mixtures.
abstract: Classic blotting methods remain a commonly applied approach to specific protein identification in gel electrophoresis of complex mixtures despite the inherent difficulty in band or spot matching due to significant variability of protein migration or localization in replicate blotting experiments. A direct application of both protein stain and protein blotting on a single membrane significantly reduces the complexity of the experiment and provides increased confidence of signal matching. Digital alignment of images acquired from both total protein stain and blotting development modes on a single membrane allows unambiguous spot or band assignments in these experiments as well as retention of quantitative information acquired from both modes of signal generation. A direct and simple method applying a fluorescent protein stain that is compatible with subsequent detection by antibody or lectin recognition factors along with common image adjustment software is examined. The utility of this blot dual-mode development method for direct protein recognition and quantification in one- and two-dimensional electrophoresis is demonstrated for bioanalytical objectives where replicate experiments are challenged by sample complexity.
authors.affiliation: Analytical Chemistry Research, Abbott Diagnostics, Abbott Laboratories, Abbott Park, IL 60064, USA. tracey.rae@abbott.com
authors.forename: Tracey
authors.initials: T
authors.lastname: Rae
authors.forename: Ryan
authors.initials: R
authors.lastname: Bonn
authors.forename: Ewa
authors.initials: E
authors.lastname: Lang
authors.forename: Svetoslava
authors.initials: S
authors.lastname: Stamenova
authors.forename: Glamarie
authors.initials: G
authors.lastname: Burgos
authors.forename: Kevin
authors.initials: K
authors.lastname: Rupprecht
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Fishpaugh
mesh_terms: D000906:Antibodies / Q000737:chemistry; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007091:Image Processing, Computer-Assisted; D037102:Lectins / Q000737:chemistry; D015336:Molecular Probe Techniques; D009942:Organometallic Compounds / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D000906:Antibodies; D037102:Lectins; D009942:Organometallic Compounds; D011506:Proteins; D011994:Recombinant Proteins; C515601:Sypro Ruby