Eastern blot

The eastern blot is a biochemical technique used to analyze protein post translational modifications (PTM) such as lipids, phosphomoieties and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, eastern blotting can be considered an extension of the biochemical technique of western blotting. Multiple techniques have been described by the term eastern blotting, most use proteins 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]

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 thereof. All of the definitions are a derivative of the technique of western blotting 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[25] has interviewed Ed Southern, originator of the Southern blot, regarding a re-christening 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

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

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 protein modifications 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 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

References

  1. 1 2 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): 4350–4. doi:10.1073/pnas.76.9.4350. PMC 411572. PMID 388439.
  3. Reinhart and Malamud; Malamud, D (1982). "Protein transfer from isoelectric focusing gels: the native blot". Analytical Biochemistry 145 (2): 229–235. doi:10.1016/0003-2697(82)90439-0. PMID 6181706.
  4. 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: 369–372. doi:10.1016/0014-5793(82)80202-0.
  5. Rocco, R.M., ed. (2005). Landmark papers in Clinical Chemistry. p. 385. ISBN 978-0-444-51950-4.
  6. Wreschner, D.H and 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.
  7. 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.
  8. 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.
  9. 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.
  10. 1 2 Ishikawa & Taki; Taki, T (2000). "Thin-layer chromatography blotting using polyvinylidene difluoride membrane (Far eastern blotting) and its applications.". Methods in Enzymology 312: 145–57. doi:10.1016/S0076-6879(00)12905-2. PMID 11070868.
  11. 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.
  12. 1 2 Tanaka; Fukuda, N; Shoyama, Y; et al. (2009). "Antigenic protein modifications in Ehrlichia". Journal of Agricultural and Food Chemistry 31 (10): 296–303. doi:10.1021/jf063457m. PMID 17455950.
  13. 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.
  14. 1 2 3 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.
  15. 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.
  16. Kurien & Scofield; Scofield, RH (2006). "Western Blotting". Methods 38 (4): 283–293. doi:10.1016/j.ymeth.2005.11.007. PMID 16483794.
  17. Buxbaum (2009). "Cationic electrophoresis and Eastern blotting". Methods in Molecular Biology 536: 115–128. doi:10.1007/978-1-59745-542-8_14. PMID 19378051.
  18. Leca-Bouvier & Blum; Blum, Loïc (2005). "Biosensors for protein detection: A review". Analytical Letters 38: 1491. doi:10.1081/AL-200065780.
  19. Jayasena (1999). "Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics". Clinical Chemistry 45 (9): 1628–1650. PMID 10471678.
  20. 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.
  21. 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.
  22. Commercially available eastern blot kits Archived September 5, 2009 at the Wayback Machine
  23. 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.
  24. 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
  25. Eastern blot on the landscape
  26. Luttman, Bratke and Kupper (2006). Immunology. Academic Press. p. 11. ISBN 978-0-12-088544-2.
  27. 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 (2): 265–270. PMC 1172571. PMID 1275889.
  28. Mann, M; Jensen, ON (2003). "Proteomic analysis of post-translational modifications". Nature Biotechnology 21 (3): 255–261. doi:10.1038/nbt0303-255. PMID 12610572.
  29. Walsh, CT; Garneau-Tsodikova, S; Gatto, GJ Jr (2005). "Protein posttranslational modifications: The chemistry of proteome diversifications". Angewandte Chemie International Edition in English 44: 7342–7372. doi:10.1002/anie.200501023. PMID 16267872.


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