Eastern+Blotting

Audrey Stryker = = =Basic Description = Eastern blotting is a technique that is used to analyze and observe protein post translational modifications (PTMs) including lipids, phosphomoieties, and glycoconjugates. It is most commonly used to identify carbohydrate epitopes. Most of the time, proteins or lipids are blotted from SDS-PAGE gel on to a nitrocellulose or PVDF membrane. These proteins are then analyzed for PTMs using a probe that is able to identify lipids and carbohydrates. Eastern blotting is similar to Western blotting; however, it is considered to be more of an extension of the Western technique (Freeze, 1993).

 Figure 1. Overview of blotting steps (Nicholas and Nelson, 2013).

= Purpose = Before becoming functional, most proteins that are translated from mRNA experience modifications known as post translational modifications (PTMs). The purpose of Eastern blotting is to examine these PTMs in proteins and lipids (Freeze, 1993). These modifications can include, but are not limited to, acetylation, alkylation, formylation, lipoylation, methylation, prenylation, and sulfation. The expression of PTMs can also be extremely useful in determining and evaluating diseases (Mann & Jensen, 2003).

=Origins = In 1982, the technique of Eastern blotting was initially rejected by scientists Michael Reinhart and Daniel Malamud who considered it to be too similar to Western blotting (Reinhart & Malamud, 1982). In 1984, a technique referred to as Middle Eastern blotting emerged. It involved the immobilization of a blot of polyA RNA which is then probed using DNA (Wreschner & Herzberg, 1984). The Eastern-Western blot was developed in 1996 and involved the blotting of phospholipids on PVDF or nitrocellulose membranes, which is the same technique as the current Eastern blot method. However, this method was used to observe specific antibodies instead of post translational modifications of proteins (Bogdanov et al., 1996). In 2001, the “original” Eastern blotting technique was used as a way to detect glycoconjugates by blotting proteins on to a PVDF membrane which is then followed by a periodate treatment. A new conjugate was created on the PVDF membrane by treating the oxidized protein with a complex mixture. The probe was then used to observe epitopes of the protein. This method was first described by Shan et al. (2001).

According to an experiment conducted by Morinaga et al. (2010), a new Eastern blotting technique was observed for ginsenoside Re (G-Re) that was found in American ginseng berry pulp extracts. The G-Re was extracted using four extracts: 100% methanol, 100% ethanol, 50% aqueous methanol, and 50% aqueous ethanol. These extracts were each applied to a polyethersulfone membrane, instead of a nitrocellulose or PVDF membrane used in traditional Eastern blotting techniques. Using this new technique, the authors were able to determine the most appropriate solvent for this technique was 50% aqueous methanol when extracting G-Re from the pulp of American ginseng berry. This is also a relatively new technique that is an extension of the traditional Eastern blotting method (Morinaga et al., 2010).
 * Recent Research **

Another experiment was conducted to establish an Eastern blotting technique that could be used to easily visualize and determine the presence of sennoside A (SA) and sennoside B (SB) in plant extracts. The SA and SB were separated and transferred to a PVDF membrane and treated with 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide hydrochloride solution followed by a bovine serum albumin treatment. Using the process of immunostaining, this style of Eastern blotting resulted in the visualization of molecular compounds. This blotting method was found to have the ability to identify SA and SB that exists within several impurities (Morinaga et al., 2009).

An experiment conducted by Tanaka et al. (2011) used Eastern blotting to develop the “fingerprint” of a natural product via monoclonal antibodies. The antibodies were separated, oxidized, and reacted to produce conjugates that can be recognized with anti-solamargine monoclonal antibodies (MAb). Fingerprinting via Eastern blotting was proven to be effective using anti-ginsenoside RB1 MAb. It was also determined that by double-staining the ginsenosides, there was potential to show the pharmacological activity of each ginsenosides (Tanaka et al., 2011).

=**References **= Bogdanov, M., Sun, J., Kaback, H.R. & Dowhan, W. (1996). A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein. Journal of Biological Chemistry, 271(20), 11615–11618.

Freeze, H.H. (1993). "Preparation and analysis of glycoconjugates". Current Protocols in Molecular Biology.

Mann, M. & Jensen, O.N. (2003). Proteomic analysis of post-translational modifications. Nature Biotechnology, 21, 255–261.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12pt;">Morinaga, O., Uto, T., Sakamoto, S., Putalun, W., Lhieochaiphant, S., Tanaka, H. & Shoyama, Y. (2009). Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-sennoside B monoclonal antibodies. Phytochemical Analysis, 20(2), 154-158.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12pt;">Morinaga, O., Uto, T., Yuan, C., Tanaka, H. & Shoyama, Y. (2010). Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts. Fitoterapia, 81(4), 284-288.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Nicholas, M.W. and Nelson, K. (2013). North, South, or East? Blotting techniques. //Journal of Investigative Dermatology, 133//, doi: 10.1038/jid.2013.216

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12pt;">Reinhart, M. & Malamud, D. (1982). Protein transfer from isoelectric focusing gels: the native blot. Analytical Biochemistry, 145(2), 229–235.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12pt;">Shan, S., Tanaka, H. & Shoyama, Y. (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), 5784–5790.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12pt;">Tanaka, H., Putalun, W. & Shoyama, Y. (2011). Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies. Chromatography Research International, 2012, 1-7.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 12pt;">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.