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.

• (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 SDS-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 PVDF or 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 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.^[11] This method has also been discussed in later work by the same group.^[12][13] The method is essentially Far-Eastern blotting.^[14]
• (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.^[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 PVDF 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 South-western blotting.
• (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 PVDF 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 South-western blotting.
• (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 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

• Far-western blotting
• Far-eastern blotting
• Glycosylation
• Phosphorylation
• Northwestern blot
• Western blot
• Northern blot
• Southern blot

References

1. 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. 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. 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. 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.