Eastern blot

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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]

Contents

History and multiple definitions

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.

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

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. 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

Related Research Articles

Molecular biology is the study of chemical and physical structure of biological macromolecules. It is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.

<span class="mw-page-title-main">Northern blot</span> Molecular biology technique

The northern blot, or RNA blot, is a technique used in molecular biology research to study gene expression by detection of RNA in a sample.

<span class="mw-page-title-main">Southern blot</span> DNA analysis technique

Southern blot is a method used for detection and quantification of a specific DNA sequence in DNA samples. This method is used in molecular biology. Briefly, purified DNA from a biological sample is digested with restriction enzymes, and the resulting DNA fragments are separated by using an electric current to move them through a sieve-like gel or matrix, which allows smaller fragments to move faster than larger fragments. The DNA fragments are transferred out of the gel or matrix onto a solid membrane, which is then exposed to a DNA probe labeled with a radioactive, fluorescent, or chemical tag. The tag allows any DNA fragments containing complementary sequences with the DNA probe sequence to be visualized within the Southern blot.

<span class="mw-page-title-main">Proteomics</span> Large-scale study of proteins

Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body.

<span class="mw-page-title-main">Western blot</span> Analytical technique used in molecular biology

The western blot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.

<span class="mw-page-title-main">Immunostaining</span> Biochemical technique

In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants won't interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<span class="mw-page-title-main">Blot (biology)</span>

A blot, in molecular biology and genetics, is a method of transferring proteins, DNA or RNA onto a carrier. In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by colorant staining, autoradiographic visualization of radiolabelled molecules, or specific labelling of some proteins or nucleic acids. The latter is done with antibodies or hybridization probes that bind only to some molecules of the blot and have an enzyme joined to them. After proper washing, this enzymatic activity is visualized by incubation with proper reactive, rendering either a colored deposit on the blot or a chemiluminescent reaction which is registered by photographic film.

A protein microarray is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. The concept and methodology of protein microarrays was first introduced and illustrated in antibody microarrays in 1983 in a scientific publication and a series of patents. The high-throughput technology behind the protein microarray was relatively easy to develop since it is based on the technology developed for DNA microarrays, which have become the most widely used microarrays.

<span class="mw-page-title-main">Southwestern blot</span> Molecular biology technique

The southwestern blot, is a lab technique that involves identifying as well as characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Determination of molecular weight of proteins binding to DNA is also made possible by the technique. The name originates from a combination of ideas underlying Southern blotting and Western blotting techniques of which they detect DNA and protein respectively. Similar to other types of blotting, proteins are separated by SDS-PAGE and are subsequently transferred to nitrocellulose membranes. Thereafter southwestern blotting begins to vary with regards to procedure as since the first blotting’s, many more have been proposed and discovered with goals of enhancing results. Former protocols were hampered by the need for large amounts of proteins and their susceptibility to degradation while being isolated.

<span class="mw-page-title-main">Ponceau S</span> Chemical compound

Ponceau S, Acid Red 112, or C.I. 27195 is a sodium salt of a diazo dye of a light red color, that may be used to prepare a stain for rapid reversible detection of protein bands on nitrocellulose or polyvinylidene fluoride (PVDF) membranes, as well as on cellulose acetate membranes. A Ponceau S stain is useful because it does not appear to have a deleterious effect on the sequencing of blotted polypeptides and is therefore one method of choice for locating polypeptides on western blots for blot-sequencing. It is also easily reversed with water washes, facilitating subsequent immunological detection. The stain can be completely removed from the protein bands by continued washing. Common stain formulations include 0.1% (w/v) Ponceau S in 5% acetic acid or 2% (w/v) Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid.

<span class="mw-page-title-main">Dot blot</span>

A dot blot is a technique in molecular biology used to detect proteins. It represents a simplification of the western blot method, with the exception that the proteins to be detected are not first separated by electrophoresis. Instead, the sample is applied directly on a membrane in a single spot, and the blotting procedure is performed.

The far-western blot, or far-western blotting, is a molecular biological method based on the technique of western blot to detect protein-protein interaction in vitro. Whereas western blot uses an antibody probe to detect a protein of interest, far-western blot uses a non-antibody probe which can bind the protein of interest. Thus, whereas western blotting is used for the detection of certain proteins, far-western blotting is employed to detect protein/protein interactions.

<span class="mw-page-title-main">Electroblotting</span>

Electroblotting is a method in molecular biology/biochemistry/immunogenetics to transfer proteins or nucleic acids onto a membrane by using PVDF or nitrocellulose, after gel electrophoresis. The protein or nucleic acid can then be further analyzed using probes such as specific antibodies, ligands like lectins, or stains. This method can be used with all polyacrylamide and agarose gels. An alternative technique for transferring proteins from a gel is capillary blotting.

The far-eastern blot, or far-eastern blotting, is a technique for the analysis of lipids separated by high-performance thin layer chromatography (HPTLC). When executing the technique, lipids are transferred from HPTLC plates to a PVDF membrane for further analysis, for example by enzymatic or ligand binding assays and mass spectrometry. It was developed in 1994 by Taki and colleagues at the Tokyo Medical and Dental University, Japan.

A reverse phase protein lysate microarray (RPMA) is a protein microarray designed as a dot-blot platform that allows measurement of protein expression levels in a large number of biological samples simultaneously in a quantitative manner when high-quality antibodies are available.

<span class="mw-page-title-main">Proximity ligation assay</span>

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The northwestern blot, also known as the northwestern assay, is a hybrid analytical technique of the western blot and the northern blot, and is used in molecular biology to detect interactions between RNA and proteins. A related technique, the western blot, is used to detect a protein of interest that involves transferring proteins that are separated by gel electrophoresis onto a nitrocellulose membrane. A colored precipitate clusters along the band on the membrane containing a particular target protein. A northern blot is a similar analytical technique that, instead of detecting a protein of interest, is used to study gene expression by detection of RNA on a similar membrane. The northwestern blot combines the two techniques, and specifically involves the identification of labeled RNA that interact with proteins that are immobilized on a similar nitrocellulose membrane.

A blotting matrix, in molecular biology and genetics, is the substrate onto which macromolecules, such as proteins, are transferred in a blot method. The matrices are generally chemically modified paper filters or microporous membrane filters. In a dot blot, macromolecules are applied directly to the matrix. Macromolecules can also be separated and transferred via gel electrophoresis.

Normalization of Western blot data is an analytical step that is performed to compare the relative abundance of a specific protein across the lanes of a blot or gel under diverse experimental treatments, or across tissues or developmental stages. The overall goal of normalization is to minimize effects arising from variations in experimental errors, such as inconsistent sample preparation, unequal sample loading across gel lanes, or uneven protein transfer, which can compromise the conclusions that can be obtained from Western blot data. Currently, there are two methods for normalizing Western blot data: (i) housekeeping protein normalization and (ii) total protein normalization.

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. S2CID   205153650.
  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. Bibcode:1979PNAS...76.4350T. 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. 123 (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 (2): 369–372. doi: 10.1016/0014-5793(82)80202-0 . S2CID   85394990.
  5. ↑ Rocco, R.M., ed. (2005). Landmark papers in Clinical Chemistry . p.  385. ISBN   978-0-444-51950-4.
  6. ↑ 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.
  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. Vol. 312. pp. 145–57. doi:10.1016/S0076-6879(00)12905-2. ISBN   9780121822132. 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. (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.
  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. S2CID   44234050.
  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". 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.
  18. ↑ Leca-Bouvier & Blum; Blum, Loïc (2005). "Biosensors for protein detection: A review". Analytical Letters. 38 (10): 1491. doi:10.1081/AL-200065780. S2CID   94503772.
  19. ↑ 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.
  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; 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.
  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. S2CID   205266061.
  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 (45): 7342–7372. doi:10.1002/anie.200501023. PMID   16267872. S2CID   32157563.
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