Eastern blot
Eastern blotting is a technique to detect protein post translational modification and is an extension of the biochemical technique of western blotting. Proteins blotted from two dimensional SDS-PAGE gel on to a PVDF or nitrocellulose membrane are analyzed for post-translational protein modifications using probes specifically designed to detect lipids, carbohydrate, phosphorylation or any other protein modification.
The technique was developed to detect protein modifications in two species of Ehrlichia- E. muris and IOE. Cholera toxin B subunit (which detects lipids), Concanavalin A (which detects glucose moieties) and nitrophospho molybdate-methyl green (detects phosphoproteins) were used to detect protein modifications. The technique showed that the antigenic proteins of the non-virulent E. muris is more post-translationally modified than the highly virulent IOE .[1]
History
The technique was conceptualized (in the East - KFRI, Kerala, India) by S. Thomas while working on sandal spike phytoplasma and developed at the Dept. of Pathology, University of Texas Medical Branch, Galveston, Texas, while working on the intracellular bacteria, Ehrlichia.
Significance
Most proteins that are translated from mRNA undergo modifications before becoming functional in cells. The modifications collectively, are known as post-translational modifications (PTMs). The nascent or folded proteins, which are stable under physiological conditions, are then subjected to a battery of specific enzyme-catalyzed modifications on the side chains or backbones.
Post-translational protein modifications can include: acetylation, acylation (myristoylation, palmitoylation), alkylation, arginylation, biotinylation, formylation, geranylgeranylation, glutamylation, glycosylation, glycylation, hydroxylation, isoprenylation, lipoylation, methylation, nitroalkylation, phosphopantetheinylation, phosphorylation, prenylation, selenation, S-nitrosylation, sulfation, transglutamination and ubiquitination (sumoylation).[2][3]
Post-translational modifications occurring at the N-terminus of the amino acid chain play an important role in translocation across biological membranes. These include secretory proteins in prokaryotes and eukaryotes and also proteins that are intended to be incorporated in various cellular and organelle membranes such as lysosomes, chloroplast, mitochondria and plasma membranes. Expression of posttranslated proteins is important in several diseases.
References
- ^ Thomas S, Thirumalapura N, Crossley EC, Ismail N, and Walker DH (2009). Antigenic protein modifications in Ehrlichia. Parasite Immunology 31: 296-303][1].
- ^ Mann M and Jensen ON (2003). Proteomic analysis of post-translational modifications. Nature Biotechnology 21, 255 - 261.
- ^ Walsh CT, Garneau-Tsodikova S, and Gatto GJ Jr. (2005). Protein posttranslational modifications: The chemistry of proteome diversifications. Angewandte Chemie International Edition in English 44,7342-7372.