Eastern blot: Difference between revisions
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'''Eastern blotting''' is a technique to detect protein [[post translational modification]]s (PTM) 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 that may detect [[lipids]], [[carbohydrate]], [[phosphorylation]] or any other protein modification. |
'''Eastern blotting''' is a technique to detect protein [[post translational modification]]s (PTM) and is an extension of the biochemical technique of [[western blotting]]. The method can be referred to as [[far-western blotting]], the detection of proteins using non-antibody proteins. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the PTM and the probe. The most common application is [[lectin]] blotting, however a variety of other probes can be used for detecting different PTMs. Proteins blotted from two dimensional [[SDS-PAGE]] gel on to a [[PVDF]] or [[nitrocellulose]] membrane are analyzed for post-translational protein modifications using probes that may detect [[lipids]], [[carbohydrate]], [[phosphorylation]] or any other protein modification. |
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==History== |
==History== |
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The first applications of Eastern blotting were the use of lectins to detect protein [[glycosylation]]. The earliest example is attributed to Tanner and Anstee in 1976, where lectins were used to detect glycosylated proteins isolated from human erythrocytes.<ref name="Tanner">{{cite journal |author=Tanner, MJ and Anstee, DJ |title=A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane |journal=Biochemistry Journal |volume=153 |pages=265-270 |year=1976 |pmid=1275889 |doi=}}</ref> The specific detection of glycosylation is often referred to as ''lectin blotting''. A summary of more recent improvements of the protocol has been provided by H. Freeze.<ref name="Freeze">{{cite journal |author=Freeze, HH |title=Preparation and analysis of glycoconjugates |journal=Current Protocols in Molecular Biology |volume= |pages=17.7.1-17.7.8 |year=1993 |pmid= |doi=10.1002/0471142727.mb1707s23}}</ref> |
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The |
The term "Eastern blotting" is used only rarely in primary literature, and has appeared more in recent publications.<ref name="Ishikawa">{{cite journal |author=Ishikawa D and Taki T |title=Microscale analysis of lipids by far-eastern blot (TLC blot) |journal=Nihon Yukagaku Kaishi |volume=47 |pages=963-970 |year=1998 |pmid= |doi=}}</ref> 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.<ref>Thomas S, Thirumalapura N, Crossley EC, Ismail N, and Walker DH (2009). Antigenic protein modifications in ''Ehrlichia''. ''Parasite Immunology'' 31, 296-303. [[http://www3.interscience.wiley.com/journal/121641379/abstract]]</ref> |
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==Significance== |
==Significance== |
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==See also== |
==See also== |
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* [[Far-western blotting]] |
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* [[Far-Eastern blotting]] |
* [[Far-Eastern blotting]] |
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* [[Glycosylation]] |
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* [[Phosphorylation]] |
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* [[Western blotting]] |
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* [[Northern blotting]] |
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* [[Southern blotting]] |
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[[Category:Carbohydrate chemistry]] |
[[Category:Carbohydrate chemistry]] |
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Revision as of 03:06, 14 June 2009
Eastern blotting is a technique to detect protein post translational modifications (PTM) and is an extension of the biochemical technique of western blotting. The method can be referred to as far-western blotting, the detection of proteins using non-antibody proteins. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the PTM and the probe. The most common application is lectin blotting, however a variety of other probes can be used for detecting different PTMs. Proteins blotted from two dimensional SDS-PAGE gel on to a PVDF or nitrocellulose membrane are analyzed for post-translational protein modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification.
History
The first applications of Eastern blotting were the use of lectins to detect protein glycosylation. The earliest example is attributed to Tanner and Anstee in 1976, where lectins were used to detect glycosylated proteins isolated from human erythrocytes.[1] The specific detection of glycosylation is often referred to as lectin blotting. A summary of more recent improvements of the protocol has been provided by H. Freeze.[2]
The term "Eastern blotting" is used only rarely in primary literature, and has appeared more in recent publications.[3] 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. 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).[5][6]
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.
References
- ^ Tanner, MJ and Anstee, DJ (1976). "A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane". Biochemistry Journal. 153: 265–270. PMID 1275889.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ Freeze, HH (1993). "Preparation and analysis of glycoconjugates". Current Protocols in Molecular Biology: 17.7.1-17.7.8. doi:10.1002/0471142727.mb1707s23.
- ^ Ishikawa D and Taki T (1998). "Microscale analysis of lipids by far-eastern blot (TLC blot)". Nihon Yukagaku Kaishi. 47: 963–970.
- ^ 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.