Applied Medical Chemistry Molecular Biochemistry 111 1702805 The Eastern Blooting Assist .Prof. Dr. Samir Ali Abd El-Kaream Collected by Safia Ali Hussein The eastern blot, or eastern blotting, is a biochemical technique used to analyze protein post-translational modifications (PTM) including the addition of lipids, phosphates, and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, eastern blot can be considered an extension of the biochemical technique of western blot. Multiple techniques have been described by the term "eastern blot(ting)", most use props protein blotted from SDS- PAGE gel on to a PVDF or nitrocellulose membrane. Transferred proteins are analyzed for post-translational modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the PTM and the probe, distinguishing them from a standard far- western blot. In principle, eastern blotting is similar to lectin blotting (i.e. detection of carbohydrate epitopes on proteins or lipids).[1] Contents definitions Applications Significance References definitions Definition of the term eastern blot is somewhat confused due to multiple sets of authors dubbing a new method as eastern blot, or a derivative thereof. All of the definitions are a derivative of the technique of western blot developed by Towbin in 1979.[2] The current definitions are summarized below in order of the first use of the name; however, all are based on some earlier works. In some cases, the technique had been in practice for some time before the introduction of the term. There is clearly no single accepted definition of the term. A recent highlight article[5] has interviewed Ed Southern, originator of the Southern blot, regarding a rechristening of eastern blotting from Tanaka et al.[3] The article likens the eastern blot to "fairies, unicorns, and a free lunch" and states that eastern blots "don't exist." The eastern blot is mentioned in an immunology textbook which compares the common blotting methods (Southern, northern and western), and states that "the eastern blot, however, exists only in test questions."[6] The principles used for eastern blotting to detect glycans can be traced back to the use of lectins to detect protein glycosylation. The earliest example for this mode of detection is Tanner and Anstee in 1976, where lectins were used to detect glycosylated proteins isolated from human erythrocytes.[7] The specific detection of glycosylation through blotting is usually referred to as lectin blotting. A summary of more recent improvements of the protocol has been provided by H. Freeze.[1] Applications One application of the technique includes detection of protein modifications in two bacterial species Ehrlichia- E. muris and IOE. Cholera toxin B subunit (which binds to gangliosides), concanavalin A (which detects mannose- containing glycans) and nitrophospho molybdate-methyl green (which 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.[4] Significance Most proteins that are translated from mRNA undergo modifications before becoming functional in cells. These modifications are collectively 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 modification of proteins can include: acetylation, acylation (myristoylation, palmitoylation), alk ylation, arginylation, ADPribosylation, biotinylation, formylation, geranylgeranylation, glutamylation, glycosylation, glycylation, hyd roxylation, isoprenylation, lipoylation, methylation, nitroalkylation , phosphopantetheinylation, phosphorylation, prenylation, selenatio n, Snitrosylation, succinylation, sulfation, transglutamination and u biquitination (sumoylation, neddylation).[8][9] 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 suchas lysosomes, chloroplast, mitochondria and plasma membrane. Expression of posttranslated proteins is important in several diseases. References 1. ^ Jump up to:a b Freeze, HH (1993). "Preparation and analysis of glycoconjugates". Current Protocols in Molecular Biology. Chapter 17: 17.7.1– 17.7.8. doi:10.1002/0471142727.mb1707s23. PMID 18265163 . 2. ^ Towbin; Staehelin, T; Gordon, J; et al. (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications". PNAS. 76(9): 4350– doi:10.1073/pnas.76.9.4350. PMC 411572. PMID 388439. 3. ^ Commercially available eastern blot kits Archived September 5, 2009, at the Wayback Machine. 4^ Mariappa D, Sauert K, Mariño K, Turnock D, Webster R, van Aalten DM, Ferguson MA, Müller HA. Protein O- GlcNAcylation is required for fibroblast growth factor signaling in Drosophila.Sci Signal. 2011 Dec 20;4(204) ra89.http://davapc1.bioch.dundee.ac.uk/pdf/nesthocker.pdf 5^ Eastern blot on the landscape 6^ Luttman, Bratke and Kupper (2006). Immunology. Academic Press. p. 11. ISBN 978-0-12-088544-2. 7^ Tanner, MJ; Anstee, DJ (1976). "A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane". Biochemical Journal. 153 (2): 265– 270. doi:10.1042/bj1530265. PMC 1172571. PMID 1275889 . 8^ Mann, M; Jensen, ON (2003). "Proteomic analysis of post-translational modifications". Nature Biotechnology. 21 (3): 255–261. doi:10.1038/nbt0303- 255. PMID 12610572. 9^ Walsh, CT; Garneau-Tsodikova, S; Gatto, GJ Jr (2005). "Protein posttranslational modifications: The chemistry of proteome diversifications". Angewandte Chemie International Edition in English. 44 (45): 7342– 7372. doi:10.1002/anie.200501023. PMID 16267872. .