Eastern blotting
Encyclopedia
Eastern blotting is a biochemical technique used to analyze protein post translational modifications (PTM) such as lipids and glycoconjugates. It is most often used to detect carbohydrate
epitope
s. 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 or lipids 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); however, the term lectin blotting is more prevalent in the literature.
There is clearly no single accepted definition of the term. A recent highlight article has interviewed Ed Southern
, originator of the Southern blot
, regarding a re-christening of Eastern blotting from Tanaka et al. 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."
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. 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.
(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.
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).
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.
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...
epitope
Epitope
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope...
s. 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 or lipids blotted from SDS-PAGE
SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
gel on to a PVDF or nitrocellulose
Nitrocellulose
Nitrocellulose is a highly flammable compound formed by nitrating cellulose through exposure to nitric acid or another powerful nitrating agent. When used as a propellant or low-order explosive, it is also known as guncotton...
membrane. Transferred proteins are analyzed for post-translational modifications using probes that may detect lipids, carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...
, phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
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
Far-western blotting
Far-Western blotting is a molecular biological method which is based on the technique of Western blotting. While usual Western blotting uses an antibody to detect a protein of interest, far-Western blotting uses a non-antibody protein, which can bind the protein of interest...
. In principle, Eastern blotting is similar to lectin
Lectin
Lectins are sugar-binding proteins that are highly specific for their sugar moieties. They play a role in biological recognition phenomena involving cells and proteins. For example, some viruses use lectins to attach themselves to the cells of the host organism during infection...
blotting (i.e. detection of carbohydrate epitopes on proteins or lipids); however, the term lectin blotting is more prevalent in the literature.
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 therof. All of the definitions are a derivative of the technique of Western blotting developed by Towbin in 1979. 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; Peferoen et al., opted to refer to their method of drawing SDS-gel separated proteins onto nitrocellulose using a vacuum as Vacuum blotting.
- (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.
- (1996) Eastern-Western blot was first used by Bogdanov et al. 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, and was based on a similar method introduced by Towbin in 1984.
- (2000) Far-Eastern blotting seems to have been first named in 2000 by Ishikawa & Taki. 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. This method has also been discussed in later work by the same group. The method is essentially Far-Eastern blotting.
- (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. Since this is essentially a Western blotWestern blotThe western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
, the charge reversal was used to dub this method an Eastern blot. - (2005) Eastern blot has been used to describe a blot of proteins on PVDF membrane where the probe is an aptamerAptamerAptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as...
rather than an antibody. This could be seen as similar to a Southern blotSouthern blotA Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
, however the interaction is between a DNA molecule(the aptamer) and a protein, rather than two DNA molecules. - (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.
- (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. These authors distinguish the method from the Far-eastern blot named by Taki et al. in that they use lectin probes and other staining reagents.
There is clearly no single accepted definition of the term. A recent highlight article has interviewed Ed Southern
Edwin Southern
Sir Edwin Mellor Southern, FRS is an English 2005 Lasker Award-winning molecular biologist. His award was for the invention of the Southern blot, now a common laboratory procedure, when he was working at the University of Edinburgh....
, originator of the Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
, regarding a re-christening of Eastern blotting from Tanaka et al. 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."
The principles used for Eastern blotting to detect glycans can be traced back to the use of lectins to detect protein glycosylation
Glycosylation
Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule . In biology glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules...
. 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. 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.
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 AConcanavalin A
Concanavalin A is a lectin originally extracted from the jack-bean, Canavalia ensiformis. It binds specifically to certain structures found in various sugars, glycoproteins, and glycolipids, mainly internal and nonreducing terminal α-D-mannosyl and α-D-glucosyl groups...
(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.
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
Acetylation
Acetylation describes a reaction that introduces an acetyl functional group into a chemical compound...
, acylation
Acylation
In chemistry, acylation is the process of adding an acyl group to a compound. The compound providing the acyl group is called the acylating agent....
(myristoylation
Myristoylation
Myristoylation is an irreversible, co-translational protein modification found in animals, plants, fungi, protozoans and viruses. In this protein modification, a myristoyl group is covalently attached via an amide bond to the alpha-amino group of an N-terminal amino acid of a nascent polypeptide...
, palmitoylation
Palmitoylation
S-Palmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to cysteine residues of membrane proteins. The precise function of palmitoylation depends on the particular protein being considered. Palmitoylation enhances the hydrophobicity of proteins and contributes to their...
), alkylation
Alkylation
Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene . Alkylating agents are widely used in chemistry because the alkyl group is probably the most common group encountered in...
, arginylation, biotinylation
Biotinylation
In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to perturb the natural function of the molecule due to the small size of biotin...
, formylation
Formylation
Formylation is a type of posttranslational modification in which a formyl group is added to the N-terminus of a protein....
, geranylgeranylation
Geranylgeranylation
Geranylgeranylation is a form of prenylation, which is a post-translational modification of proteins that involves the attachment of one or two 20-carbon lipophilic geranylgeranyl isoprene units from geranylgeranyl diphosphate to one or two cysteine residue at the C-terminus of specific proteins...
, glutamylation, glycosylation
Glycosylation
Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule . In biology glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules...
, glycylation, hydroxylation
Hydroxylation
Hydroxylation is a chemical process that introduces a hydroxyl group into an organic compound. In biochemistry, hydroxylation reactions are often facilitated by enzymes called hydroxylases. Hydroxylation is the first step in the oxidative degradation of organic compounds in air...
, isoprenylation, lipoylation, methylation
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
, nitroalkylation, phosphopantetheinylation, phosphorylation
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
, prenylation
Prenylation
Prenylation, or isoprenylation, or lipidation is the addition of hydrophobic molecules to a protein. It is usually assumed that prenyl groups facilitate attachment to cell membranes, similar to lipid anchor like the GPI anchor, though direct evidence is missing...
, selenation, S-nitrosylation, sulfation
Sulfation
Sulfation in biochemistry is the enzyme-catalyzed addition of sulfate to another molecule. It often refers to a phase II enzyme reaction. This biotransformation process uses its cosubstrate 3'-phosphoadenosine-5'-phosphosulfate to transfer sulfate to a xenobiotic...
, transglutamination and ubiquitination (sumoylation).
Post-translational modifications occurring at the N-terminus of the amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
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
Chloroplast
Chloroplasts are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis.Chloroplasts are green...
, mitochondria and plasma membrane. Expression of posttranslated proteins is important in several diseases.
See also
- Far-western blottingFar-western blottingFar-Western blotting is a molecular biological method which is based on the technique of Western blotting. While usual Western blotting uses an antibody to detect a protein of interest, far-Western blotting uses a non-antibody protein, which can bind the protein of interest...
- Far-Eastern blotting
- GlycosylationGlycosylationGlycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule . In biology glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules...
- PhosphorylationPhosphorylationPhosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
- Western blotting
- Northern blotting
- Southern blotting