Eastern blotting is a technique to analyze proteins, lipids, or glycoconjugates, and is most often used to detect carbohydrate epitopes. Thus, Eastern blotting can be considered an extension of the biochemical technique of western blotting which detects protein post translational modifications (PTM). Multiple techniques have been described by the term Eastern blotting, most use proteins or lipids 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); however, the term lectin blotting is more prevalent in the literature.
History and multiple definitions
Definition of the term Eastern blotting is somewhat confused due to multiple sets of authors dubbing a new method as Eastern blotting, or a derivative therof. All of the definitions are a derivative of the technique of Western blotting developed by Towbin in 1979. 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.
* (1982) The term Eastern blotting was specifically rejected by two separate groups: Reinhart and Malamud referred to a protein blot of a native gel as a native blot; Peferoen et al., opted to refer to their method of drawing SDS-gel separated proteins onto nitrocellulose using a vacuum as Vacuum blotting.
* (1984) Middle Eastern blotting has been described as a blot of polyA RNA (resolved by agarose) which is then immobilized. The immobilized RNA is then probed using DNA.
* (1996) Eastern-Western blot was first used by Bogdanov et al. The method involved blotting of phospholipids on PVDF membrane, and probing with antibodies that recognized carbohydrate epitopes. This method is based on earlier work by Taki et al. in 1994, which they originally dubbed TLC blotting, and was based on a similar method introduced by Towbin in 1984.
* (2000) Far-Eastern blotting seems to have been first named in 2000 by Ishikawa & Taki. The method is described more fully in the article on Far-Eastern blotting, but is based on antibody or lectin staining of lipids transferred to PVDF membranes.
* (2001) Eastern blotting was described as a technique for detecting glycoconjugates generated by blotting BSA onto PVDF membranes, followed by periodate treatment. The oxidized protein is then treated with a complex mixture, generating a new conjugate on the membrane. The membrane is then probed with antibodies for epitopes of interest. This method has also been discussed in later work by the same group.. The method is essentially Far-Eastern blotting.
* (2002) Eastern blot has also been used to describe an immunoblot performed on proteins blotted to a PVDF membrane from a PAGE gel run with opposite polarity. Since this is essentially a Western blot, the charge reversal was used to dub this method an Eastern blot.
* (2005) Eastern blot has been used to describe a blot of proteins on PVDF membrane where the probe is an aptamer rather than an antibody. This could be seen as similar to a Southern blot, however the interaction is between a DNA molecule(the aptamer) and a protein, rather than two DNA molecules.
* (2006) Eastern blotting has been used to refer to the detection of fusion proteins through complementation. The name is based on the use of an enzyme activator (EA) as part of the detection.
* (2009) Eastern blotting has most recently been re-dubbed by Thomas et al. as a technique which probes proteins blotted to PVDF membrane with lectins, cholera toxin and chemical stains to detect glycosylated, lipoylated or phosphorylated proteins. These authors distinguish the method from the Far-eastern blot named by Taki et al. in that they use lectin probes and other staining reagents.
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. 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.
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.
Most proteins that are translated from mRNA undergo modifications before becoming functional in cells. The 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 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).
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 membrane. Expression of posttranslated proteins is important in several diseases.
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22. ^ Commercially available EAstern Blot kits
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* Far-western blotting
* Far-Eastern blotting
* Western blotting
* Northern blotting
* Southern blotting
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