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{{Short description|Biochemical technique}} |
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The '''eastern blot''' is a biochemical technique used to analyze protein [[post |
The '''eastern blot''', or '''eastern blotting''', is a biochemical technique used to analyze protein [[post-translational modification]]s including the addition of lipids, phosphates, and glycoconjugates. It is most often used to detect [[carbohydrate]] [[epitope]]s. 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 [[phosphoprotein]] blotted from [[SDS-PAGE|sodium dodecyl sulfate–polyacrylamide gel electrophoresis]] (SDS-PAGE) gel on to a [[polyvinylidene fluoride]] 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 post-translational modifications 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).<ref name="Freeze"/> |
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==History and multiple definitions== |
==History and multiple definitions== |
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Definition of the term ''eastern |
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.<ref name="towbin">{{cite journal |
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|author=Towbin |
|author=Towbin |
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|title=Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications |
|title=Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications |
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|first3=J |
|first3=J |
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|issue=9 |
|issue=9 |
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|pmc=411572|bibcode=1979PNAS...76.4350T |
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⚫ | |||
|display-authors=etal|doi-access=free |
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⚫ | |||
*(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'';<ref>{{cite journal |
*(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'';<ref>{{cite journal |
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|author=Reinhart and Malamud |
|author=Reinhart and Malamud |
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|first2=D |
|first2=D |
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|issue=2 |
|issue=2 |
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|doi=10.1016/0003-2697(82)90439-0}}</ref> Peferoen et al., opted to refer to their method of drawing |
|doi=10.1016/0003-2697(82)90439-0}}</ref> Peferoen et al., opted to refer to their method of drawing sodium dodecyl sulfate-gel separated proteins onto nitrocellulose using a vacuum as ''Vacuum blotting''.<ref>{{cite journal |
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|author=Peferoen |
|author=Peferoen |
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|title=Vacuum-blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose |
|title=Vacuum-blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose |
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|journal= FEBS Letters |
|journal= FEBS Letters |
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|volume= 145 |
|volume= 145 |
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|issue=2 |
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|pages= 369–372 |
|pages= 369–372 |
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|year= 1982 |
|year= 1982 |
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|doi=10.1016/0014-5793(82)80202-0|s2cid=85394990 |
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|display-authors=etal|doi-access=free |
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|bibcode=1982FEBSL.145..369P |
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}}</ref><ref>{{cite book |
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|isbn=978-0-444-51950-4 |
|isbn=978-0-444-51950-4 |
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|page=[https://archive.org/details/landmarkpaperscl00rocc/page/n402 385] |
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|page=385 |
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|title=Landmark papers in Clinical Chemistry |
|title=Landmark papers in Clinical Chemistry |
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|url=https://archive.org/details/landmarkpaperscl00rocc |
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|url-access=limited |
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|editor=Rocco, R.M. |
|editor=Rocco, R.M. |
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|year=2005 |
|year=2005|publisher=Elsevier Science |
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}}</ref> |
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*(1984) ''Middle |
*(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.<ref>{{cite journal |
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|author1=Wreschner, D.H |author2=Herzberg, M. |
|author1=Wreschner, D.H. |author2=Herzberg, M. |
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|year=1984 |
|year=1984 |
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|journal=Nucleic Acids Research |
|journal=Nucleic Acids Research |
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|last4=Dowhan |
|last4=Dowhan |
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|first4=W |
|first4=W |
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|issue=20|display-authors=etal}}</ref> The method involved blotting of phospholipids on |
|issue=20|display-authors=etal|doi-access=free}}</ref> The method involved blotting of phospholipids on polyvinylidene fluorideor nitrocellulose membrane prior to transfer of proteins onto the same nitrocellulose membrane by conventional western blotting and probing with conformation specific antibodies. This method is based on earlier work by Taki et al. in 1994, which they originally dubbed ''TLC blotting'',<ref name="taki1994">{{cite journal |
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|author=Taki |
|author=Taki |
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|title=Blotting of glycolipids and phospholipids from a high-performance thin-layer chromatogram to a polyvinylidene difluoride membrane |
|title=Blotting of glycolipids and phospholipids from a high-performance thin-layer chromatogram to a polyvinylidene difluoride membrane |
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|first5=G |
|first5=G |
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|issue=2|display-authors=etal}}</ref> |
|issue=2|display-authors=etal}}</ref> |
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*(2000) ''Far-eastern blotting'' seems to have been first named in 2000 by Ishikawa & Taki.<ref name="taki2000">{{cite |
*(2000) ''Far-eastern blotting'' seems to have been first named in 2000 by Ishikawa & Taki.<ref name="taki2000">{{cite book |
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|author=Ishikawa & Taki |
|author=Ishikawa & Taki |
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|title=Sphingolipid Metabolism and Cell Signaling, Part B |
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|title=Thin-layer chromatography blotting using polyvinylidene difluoride membrane (Far eastern blotting) and its applications. |
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| |
|series= Methods in Enzymology |
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|volume= 312 |
|volume= 312 |
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|pages= 145–57 |
|pages= 145–57 |
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Line 104: | Line 114: | ||
|pmid= 11070868|doi=10.1016/S0076-6879(00)12905-2 |
|pmid= 11070868|doi=10.1016/S0076-6879(00)12905-2 |
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|last2=Taki |
|last2=Taki |
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|first2=T|chapter=Thin-Layer Chromatography Blotting Using Polyvinylidene Difluoride Membrane (Far-Eastern Blotting) and Its Applications |
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⚫ | |||
|isbn=9780121822132 |
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⚫ | *(2001) ''Eastern blotting'' was described as a technique for detecting glycoconjugates generated by blotting BSA onto |
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⚫ | |||
⚫ | *(2001) ''Eastern blotting'' was described as a technique for detecting glycoconjugates generated by blotting BSA onto polyvinylidene fluoride 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.<ref name="shan">{{cite journal |
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|author=Shan |
|author=Shan |
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|title=Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and a new eastern blotting for glucuronides of glycyrrhetinic acid |
|title=Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and a new eastern blotting for glucuronides of glycyrrhetinic acid |
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|pages= 21–27 |
|pages= 21–27 |
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|year= 2006 |
|year= 2006 |
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|doi=10.1007/s11418-005-0005-3 |
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|last2=Shan |
|last2=Shan |
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|first2=Shaojie |
|first2=Shaojie |
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|first3=Hiroyuki |
|first3=Hiroyuki |
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|last4=Shoyama |
|last4=Shoyama |
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|first4=Yukihiro|display-authors=etal}}</ref> The method is essentially |
|first4=Yukihiro|s2cid=44234050 |
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|display-authors=etal}}</ref> The method is essentially far-eastern blot.<ref name="thomas">{{cite journal |
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|author=Thomas |
|author=Thomas |
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|title=Antigenic protein modifications in Ehrlichia |
|title=Antigenic protein modifications in Ehrlichia |
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|first5=DH |
|first5=DH |
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|issue=6 |
|issue=6 |
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|pmc=2731653|display-authors=etal}}</ref> |
|pmc=2731653|display-authors=etal}}</ref> |
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*(2002) ''Eastern blot'' has also been used to describe an immunoblot performed on proteins blotted to a |
*(2002) ''Eastern blot'' has also been used to describe an immunoblot performed on proteins blotted to a polyvinylidene fluoride membrane from a PAGE gel run with opposite polarity.<ref name="buxbaum">{{cite journal |
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|author=Buxbaum |
|author=Buxbaum |
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|title=Cationic electrophoresis and electrotransfer of membrane glycoproteins |
|title=Cationic electrophoresis and electrotransfer of membrane glycoproteins |
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|last2=Scofield |
|last2=Scofield |
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|first2=RH |
|first2=RH |
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|issue=4}}</ref><ref>{{cite |
|issue=4}}</ref><ref>{{cite book |
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|author=Buxbaum |
|author=Buxbaum |
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|title= |
|title=Protein Blotting and Detection |
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|chapter=Cationic electrophoresis and Eastern blotting |
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| |
|series=Methods in Molecular Biology |
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|volume=536 |
|volume=536 |
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|pages=115–128 |
|pages=115–128 |
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|pmid=19378051 |
|pmid=19378051 |
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|doi=10.1007/978-1-59745-542-8_14 |
|doi=10.1007/978-1-59745-542-8_14 |
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|isbn=978-1-934115-73-2 |
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}}</ref> |
}}</ref> |
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*(2005) ''Eastern blot'' has been used to describe a blot of proteins on |
*(2005) ''Eastern blot'' has been used to describe a blot of proteins on polyvinylidene fluoride membrane where the probe is an [[aptamer]] rather than an antibody.<ref name="leca-bouvier">{{cite journal |
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|author=Leca-Bouvier & Blum |
|author=Leca-Bouvier & Blum |
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|title=Biosensors for protein detection: A review |
|title=Biosensors for protein detection: A review |
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|journal= Analytical Letters |
|journal= Analytical Letters |
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|volume= 38 |
|volume= 38 |
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|issue=10 |
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|page= 1491 |
|page= 1491 |
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|year= 2005 |
|year= 2005 |
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|doi=10.1081/AL-200065780 |
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|last2=Blum |
|last2=Blum |
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|first2=Loïc|s2cid=94503772 |
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}}</ref> 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.<ref name="jayasewa">{{cite journal |
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|author=Jayasena |
|author=Jayasena |
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|title=Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics |
|title=Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics |
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|pages= 1628–1650 |
|pages= 1628–1650 |
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|year= 1999 |
|year= 1999 |
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|pmid= 10471678|doi= |
|pmid= 10471678|doi= 10.1093/clinchem/45.9.1628 |
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|issue=9|doi-access= free |
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}}</ref> The method is similar to [[southwestern blot]]. |
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*(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.<ref>{{cite journal |
*(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.<ref>{{cite journal |
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|author=Horecka |
|author=Horecka |
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|title=A novel antibody-free method for protein blotting using enzyme fragment complementation |
|title=A novel antibody-free method for protein blotting using enzyme fragment complementation |
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|journal= |
|journal=BioTechniques |
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|volume=40 |
|volume=40 |
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|year=2006 |
|year=2006 |
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|last7=Eglen |
|last7=Eglen |
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|first7=RM |
|first7=RM |
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|issue=3|display-authors=etal}}</ref><ref>{{cite journal |
|issue=3|display-authors=etal|doi-access=free |
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}}</ref><ref>{{cite journal |
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|author=Olson and Eglen |
|author=Olson and Eglen |
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|title=beta Galactosidase complementation: A cell-based luminescent assay platform for drug discovery |
|title=beta Galactosidase complementation: A cell-based luminescent assay platform for drug discovery |
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|first2=RM |
|first2=RM |
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|issue=1}}</ref><ref>[http://www.discoverx.com/additional_products/eastern_blot.php Commercially available eastern blot kits] {{webarchive |url=https://web.archive.org/web/20090905131603/http://www.discoverx.com/additional_products/eastern_blot.php |date=September 5, 2009 }}</ref> |
|issue=1}}</ref><ref>[http://www.discoverx.com/additional_products/eastern_blot.php Commercially available eastern blot kits] {{webarchive |url=https://web.archive.org/web/20090905131603/http://www.discoverx.com/additional_products/eastern_blot.php |date=September 5, 2009 }}</ref> |
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*(2009) ''Eastern blotting'' has most recently been re-dubbed by Thomas et al. as a technique which probes proteins blotted to |
*(2009) ''Eastern blotting'' has most recently been re-dubbed by Thomas et al. as a technique which probes proteins blotted to polyvinylidene fluoride membrane with lectins, cholera toxin and chemical stains to detect glycosylated, lipoylated or phosphorylated proteins.<ref name="thomas"/> These authors distinguish the method from the ''[[far-eastern blot]]'' named by Taki et al.<ref name="taki2000"/> in that they use lectin probes and other staining reagents. |
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*(2009) ''Eastern blot'' has been used to describe a blot of proteins on nitrocellulose membrane where the probe is an [[aptamer]] rather than an antibody.<ref name="Lin">{{cite journal |
*(2009) ''Eastern blot'' has been used to describe a blot of proteins on nitrocellulose membrane where the probe is an [[aptamer]] rather than an antibody.<ref name="Lin">{{cite journal |
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|author=Lin & McNatty |
|author=Lin & McNatty |
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|first2=JS |
|first2=JS |
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|issue=9 |
|issue=9 |
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|doi=10.1373/clinchem.2009.127266}}</ref> The method is similar to |
|doi=10.1373/clinchem.2009.127266|doi-access=free |
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}}</ref> The method is similar to southwestern blot. |
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*(2011) A recent study used the term eastern blotting to describe detection of glycoproteins with lectins such as concanavalin A |
*(2011) A recent study used the term eastern blotting to describe detection of glycoproteins with lectins such as concanavalin A<ref>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</ref> |
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There is clearly no single accepted definition of the term. A recent highlight article<ref>[http://www.rsc.org/chemistryworld/News/2007/May/04050701.asp Eastern blot on the landscape<!-- Bot generated title -->]</ref> has interviewed [[Edwin Southern|Ed Southern]], originator of the [[Southern blot]], regarding a |
There is clearly no single accepted definition of the term. A recent highlight article<ref>[http://www.rsc.org/chemistryworld/News/2007/May/04050701.asp Eastern blot on the landscape<!-- Bot generated title -->]</ref> has interviewed [[Edwin Southern|Ed Southern]], originator of the [[Southern blot]], regarding a rechristening of ''eastern blotting'' from Tanaka et al.<ref name="tanaka"/> 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 blot|northern]] and western), and states that "the eastern blot, however, exists only in test questions."<ref>{{cite book |
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|author=Luttman, Bratke and Kupper |
|author=Luttman, Bratke and Kupper |
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|title=Immunology |
|title=Immunology |
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|publisher=Academic Press |
|publisher=Academic Press |
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|page=11 |
|page=11 |
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| |
|isbn=978-0-12-088544-2}} |
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</ref> |
</ref> |
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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]].<ref name="Tanner">{{cite journal |author1=Tanner, MJ |author2=Anstee, DJ |title=A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane |journal=Biochemical Journal |volume=153 |pages=265–270 |year=1976 |pmid=1275889 |doi= |issue=2 |pmc=1172571}}</ref> 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.<ref name="Freeze">{{cite journal |author=Freeze, HH |title=Preparation and analysis of glycoconjugates |journal= |
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]].<ref name="Tanner">{{cite journal |author1=Tanner, MJ |author2=Anstee, DJ |title=A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane |journal=Biochemical Journal |volume=153 |pages=265–270 |year=1976 |pmid=1275889 |doi= 10.1042/bj1530265|issue=2 |pmc=1172571}}</ref> 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.<ref name="Freeze">{{cite journal |author=Freeze, HH |title=Preparation and analysis of glycoconjugates |journal=Current Protocols in Molecular Biology |volume= Chapter 17|pages=17.7.1–17.7.8 |year=1993 |pmid= 18265163|doi=10.1002/0471142727.mb1707s23|s2cid=205153650 }}</ref> |
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==Applications== |
==Applications== |
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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]]), [[ |
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 name="thomas"/> |
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==Significance== |
==Significance== |
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Most [[proteins]] that are translated from [[mRNA]] undergo modifications before becoming functional in cells. These modifications are collectively known as [[post-translational modifications]] |
Most [[proteins]] that are translated from [[mRNA]] undergo modifications before becoming functional in cells. These modifications are collectively known as [[post-translational modifications]]. 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. |
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[[Post-translational modification]] of proteins can include |
[[Post-translational modification]] of proteins can include [[acetylation]], [[acylation]] ([[myristoylation]], [[palmitoylation]]), [[alkylation]], [[arginylation]], [[ADP-ribosylation]], [[biotinylation]], [[formylation]], [[geranylgeranylation]], [[glutamylation]], [[glycosylation]], [[glycylation]], [[hydroxylation]], [[isoprenylation]], [[lipoylation]], [[methylation]], [[nitroalkylation]], [[phosphopantetheinylation]], [[phosphorylation]], [[prenylation]], [[selenation]], [[S-nitrosylation]], [[succinylation]], [[sulfation]], [[transglutamination]], [[sulfinylation]], [[sulfonylation]] and [[ubiquitination]] (sumoylation, neddylation).<ref>{{cite journal | last1 = Mann | first1 = M | last2 = Jensen | first2 = ON | year = 2003 | title = Proteomic analysis of post-translational modifications | journal = Nature Biotechnology | volume = 21 | issue = 3| pages = 255–261 |pmid=12610572 | doi=10.1038/nbt0303-255| s2cid = 205266061 }}</ref><ref>{{cite journal | last1 = Walsh | first1 = CT | last2 = Garneau-Tsodikova | first2 = S |author2-link= Sylvie Garneau-Tsodikova | last3 = Gatto | first3 = GJ Jr | year = 2005 | title = Protein posttranslational modifications: The chemistry of proteome diversifications | journal = Angewandte Chemie International Edition in English | volume = 44 | issue = 45| pages = 7342–7372 |pmid=16267872|doi=10.1002/anie.200501023| s2cid = 32157563 }}</ref> |
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[[Post-translational modification]]s 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. |
[[Post-translational modification]]s 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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==See also== |
==See also== |
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⚫ | |||
* [[Far-western blotting]] |
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* [[Far-eastern blotting]] |
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* [[Glycosylation]] |
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* [[Phosphorylation]] |
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* [[Northwestern blot]] |
* [[Northwestern blot]] |
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* [[ |
* [[Far-eastern blot]] |
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* [[ |
* [[Blot (biology)|Blot]] |
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⚫ | |||
==References== |
==References== |
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{{ |
{{Reflist|2}} |
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{{Protein methods}} |
{{Protein methods}} |
Latest revision as of 01:17, 22 June 2024
The eastern blot, or eastern blotting, is a biochemical technique used to analyze protein post-translational modifications 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 phosphoprotein blotted from sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel on to a polyvinylidene fluoride 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 post-translational modifications 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]
History and multiple definitions
[edit]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.
- (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;[3] Peferoen et al., opted to refer to their method of drawing sodium dodecyl sulfate-gel separated proteins onto nitrocellulose using a vacuum as Vacuum blotting.[4][5]
- (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.[6]
- (1996) Eastern-western blot was first used by Bogdanov et al.[7] The method involved blotting of phospholipids on polyvinylidene fluorideor nitrocellulose membrane prior to transfer of proteins onto the same nitrocellulose membrane by conventional western blotting and probing with conformation specific antibodies. This method is based on earlier work by Taki et al. in 1994, which they originally dubbed TLC blotting,[8] and was based on a similar method introduced by Towbin in 1984.[9]
- (2000) Far-eastern blotting seems to have been first named in 2000 by Ishikawa & Taki.[10] The method is described more fully in the article on far-eastern blot, but is based on antibody or lectin staining of lipids transferred to polyvinylidene fluoride membranes.
- (2001) Eastern blotting was described as a technique for detecting glycoconjugates generated by blotting BSA onto polyvinylidene fluoride 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.[11] This method has also been discussed in later work by the same group.[12][13] The method is essentially far-eastern blot.[14]
- (2002) Eastern blot has also been used to describe an immunoblot performed on proteins blotted to a polyvinylidene fluoride membrane from a PAGE gel run with opposite polarity.[15] Since this is essentially a western blot, the charge reversal was used to dub this method an eastern blot.[16][17]
- (2005) Eastern blot has been used to describe a blot of proteins on polyvinylidene fluoride membrane where the probe is an aptamer rather than an antibody.[18] 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.[19] The method is similar to southwestern blot.
- (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.[20][21][22]
- (2009) Eastern blotting has most recently been re-dubbed by Thomas et al. as a technique which probes proteins blotted to polyvinylidene fluoride membrane with lectins, cholera toxin and chemical stains to detect glycosylated, lipoylated or phosphorylated proteins.[14] These authors distinguish the method from the far-eastern blot named by Taki et al.[10] in that they use lectin probes and other staining reagents.
- (2009) Eastern blot has been used to describe a blot of proteins on nitrocellulose membrane where the probe is an aptamer rather than an antibody.[23] The method is similar to southwestern blot.
- (2011) A recent study used the term eastern blotting to describe detection of glycoproteins with lectins such as concanavalin A[24]
There is clearly no single accepted definition of the term. A recent highlight article[25] has interviewed Ed Southern, originator of the Southern blot, regarding a rechristening of eastern blotting from Tanaka et al.[12] 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."[26]
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.[27] 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
[edit]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.[14]
Significance
[edit]Most proteins that are translated from mRNA undergo modifications before becoming functional in cells. These modifications are collectively known as post-translational modifications. 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), alkylation, arginylation, ADP-ribosylation, biotinylation, formylation, geranylgeranylation, glutamylation, glycosylation, glycylation, hydroxylation, isoprenylation, lipoylation, methylation, nitroalkylation, phosphopantetheinylation, phosphorylation, prenylation, selenation, S-nitrosylation, succinylation, sulfation, transglutamination, sulfinylation, sulfonylation and ubiquitination (sumoylation, neddylation).[28][29]
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.
See also
[edit]References
[edit]- ^ 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. S2CID 205153650.
- ^ 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): 4350–4. Bibcode:1979PNAS...76.4350T. doi:10.1073/pnas.76.9.4350. PMC 411572. PMID 388439.
- ^ Reinhart and Malamud; Malamud, D (1982). "Protein transfer from isoelectric focusing gels: the native blot". Analytical Biochemistry. 123 (2): 229–235. doi:10.1016/0003-2697(82)90439-0. PMID 6181706.
- ^ Peferoen; et al. (1982). "Vacuum-blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose". FEBS Letters. 145 (2): 369–372. Bibcode:1982FEBSL.145..369P. doi:10.1016/0014-5793(82)80202-0. S2CID 85394990.
- ^ Rocco, R.M., ed. (2005). Landmark papers in Clinical Chemistry. Elsevier Science. p. 385. ISBN 978-0-444-51950-4.
- ^ Wreschner, D.H.; Herzberg, M. (1984). "A new blotting medium for the simple isolation and identification of highly resolved messenger RNA". Nucleic Acids Research. 12 (3): 1349–1359. doi:10.1093/nar/12.3.1349. PMC 318581. PMID 6701087.
- ^ Bogdanov; Sun, J; Kaback, HR; Dowhan, W; et al. (1996). "A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein". Journal of Biological Chemistry. 271 (20): 11615–11618. doi:10.1074/jbc.271.20.11615. PMID 8662750.
- ^ Taki; Handa, S; Ishikawa, D; et al. (1994). "Blotting of glycolipids and phospholipids from a high-performance thin-layer chromatogram to a polyvinylidene difluoride membrane". Analytical Biochemistry. 221 (2): 312–316. doi:10.1006/abio.1994.1418. PMID 7810872.
- ^ Towbin; Schoenenberger, C; Ball, R; Braun, DG; Rosenfelder, G; et al. (1984). "Glycosphingolipid-blotting: an immunological detection procedure after separation by thin layer chromatography". Journal of Immunological Methods. 72 (2): 471–9. doi:10.1016/0022-1759(84)90015-2. PMID 6381603.
- ^ a b Ishikawa & Taki; Taki, T (2000). "Thin-Layer Chromatography Blotting Using Polyvinylidene Difluoride Membrane (Far-Eastern Blotting) and Its Applications". Sphingolipid Metabolism and Cell Signaling, Part B. Methods in Enzymology. Vol. 312. pp. 145–57. doi:10.1016/S0076-6879(00)12905-2. ISBN 9780121822132. PMID 11070868.
- ^ Shan; Tanaka, H; Shoyama, Y; et al. (2001). "Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and a new eastern blotting for glucuronides of glycyrrhetinic acid". Analytical Chemistry. 73 (24): 5784–90. doi:10.1021/ac0106997. PMID 11791545.
- ^ a b Tanaka; Fukuda, N; Shoyama, Y; et al. (2007). "Eastern blotting and immunoaffinity concentration using monoclonal antibody for ginseng saponins in the field of traditional chinese medicines". Journal of Agricultural and Food Chemistry. 55 (10): 3783–7. doi:10.1021/jf063457m. PMID 17455950.
- ^ Fukuda; Shan, Shaojie; Tanaka, Hiroyuki; Shoyama, Yukihiro; et al. (2006). "New staining methodology: Eastern blotting for glycosides in the field of Kampo medicines". Journal of Natural Medicines. 60: 21–27. doi:10.1007/s11418-005-0005-3. S2CID 44234050.
- ^ a b c Thomas; Thirumalapura, N; Crossley, EC; Ismail, N; Walker, DH; et al. (2009). "Antigenic protein modifications in Ehrlichia". Parasite Immunology. 31 (6): 296–303. doi:10.1111/j.1365-3024.2009.01099.x. PMC 2731653. PMID 19493209.
- ^ Buxbaum; et al. (2002). "Cationic electrophoresis and electrotransfer of membrane glycoproteins". Analytical Biochemistry. 314 (1): 70–76. doi:10.1016/S0003-2697(02)00639-5. PMID 12633604.
- ^ Kurien & Scofield; Scofield, RH (2006). "Western Blotting". Methods. 38 (4): 283–293. doi:10.1016/j.ymeth.2005.11.007. PMID 16483794.
- ^ Buxbaum (2009). "Cationic electrophoresis and Eastern blotting". Protein Blotting and Detection. Methods in Molecular Biology. Vol. 536. pp. 115–128. doi:10.1007/978-1-59745-542-8_14. ISBN 978-1-934115-73-2. PMID 19378051.
- ^ Leca-Bouvier & Blum; Blum, Loïc (2005). "Biosensors for protein detection: A review". Analytical Letters. 38 (10): 1491. doi:10.1081/AL-200065780. S2CID 94503772.
- ^ Jayasena (1999). "Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics". Clinical Chemistry. 45 (9): 1628–1650. doi:10.1093/clinchem/45.9.1628. PMID 10471678.
- ^ Horecka; Charter, NW; Bosano, BL; Fung, P; Kobel, P; Peng, K; Eglen, RM; et al. (2006). "A novel antibody-free method for protein blotting using enzyme fragment complementation". BioTechniques. 40 (3): 381–383. doi:10.2144/000112119. PMID 16568826.
- ^ Olson and Eglen; Eglen, RM (2007). "beta Galactosidase complementation: A cell-based luminescent assay platform for drug discovery". ASSAY and Drug Development Technologies. 5 (1): 137–144. doi:10.1089/adt.2006.052. PMID 17355206.
- ^ Commercially available eastern blot kits Archived September 5, 2009, at the Wayback Machine
- ^ Lin & McNatty; Lin, JS (2009). "Aptamer-Based Regionally Protected PCR for Protein Detection". Clinical Chemistry. 55 (9): 1687–1693. doi:10.1373/clinchem.2009.127266. PMID 19589846.
- ^ 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
- ^ Eastern blot on the landscape
- ^ Luttman, Bratke and Kupper (2006). Immunology. Academic Press. p. 11. ISBN 978-0-12-088544-2.
- ^ 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.
- ^ Mann, M; Jensen, ON (2003). "Proteomic analysis of post-translational modifications". Nature Biotechnology. 21 (3): 255–261. doi:10.1038/nbt0303-255. PMID 12610572. S2CID 205266061.
- ^ 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. S2CID 32157563.