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Eastern blot on the landscape


04 May 2007

Updated 09 May 2007

 

Anyone with an interest in molecular biology will probably have heard of Southern blots, northern blots and western blots, used to analyse DNA, RNA and proteins, respectively. Eastern blots, on the other hand, will be found ranked alongside fairies, unicorns, and a free lunch. They don't exist. Or rather, they didn't until Yukihiro Shoyama and colleagues at Kyushu University, Japan, claimed to have developed one.

Southern blots were named after Ed Southern, at the University of Oxford, who developed them in the 1970s.   The technique kicks off with an enzyme to chop DNA into smaller fragments; the fragments are passed through an agarose gel; the gel is treated with a solution that splits the double-stranded DNA fragments into single strands; the single stranded fragments are transferred onto a sheet of nitrocellulose; and the nitrocellulose is probed with complementary single-stranded DNA fragments. A similar technique was developed a few years later to analyse RNA, and so - with the hilarious wordplay too good to miss - the northern blot was born (small 'n' since it wasn't named after Northern, or any researcher).

In the 1980s, the technique was evolved further to detect proteins. This time, antibodies, rather than complementary strands, are used to probe the nitrocellulose sheet for proteins of interest. This became known as a western blot, but is sometimes called an immunoblot.

So what could an eastern blot possibly be used for?   Nothing, according to the consensus of internet bloggers.

But Shoyama's team beg to differ: they investigated the small molecules in ginseng, a key component of traditional Chinese medicine, with something they call an eastern blot - the technique adapted, they say, from the western blot.

In a western blot, an electric current can be used to pull separated proteins from gel to membrane, but that doesn't work for small molecules; nor is it easy to probe small molecules (unlike large proteins) on a membrane. 

So Shoyama's team instead separated ginseng saponins (ginsenosides) with thin layer chromatography (TLC). Shoyama's eastern blot technique then requires the TLC plate to be blotted to a polyvinylidene difluoride membrane, and treated with the protein bovine serum albumin, resulting in a ginsenoside-protein conjugate fixed on the membrane: large enough for the small molecules to be detected when stained, western style, with anti-ginsenoside antibodies.

It's all a bit much for Derek Lowe, a medicinal chemist experienced in preclinical drug discovery. Lowe, who writes a monthly column for Chemistry World, had wondered when somebody was going to develop an eastern blot.

'I don't think these guys have a chance of making this nomenclature catch on,' he sighed.

'There's already a good analytical method for quantitative separation of small molecules,' he continued, 'namely liquid chromatography allied with mass spectrometry (LC/MS)'. Curiously, Lowe noted, Shoyama's paper made no reference to the technique. 'If you look through the natural product literature, that's really the standard, and rightly so.' 

Further, he added, having to conjugate the small molecule to a protein isn't going to suit every small molecule. In fact, Lowe has quite a list of drawbacks to the technique.

'These are all rather ancient history, though,' he told Chemistry World, 'since quantitative TLC has long been displaced by LC/MS.'

But the last word must surely go to Ed Southern, arguably the father of the so-called compass point blots. 'I've never played any part in naming the various quarters of the blotting compass - indeed,  I wasn't keen on "blot"!' he exclaimed, when Chemistry World tracked him down. 'But eastern is a gap; and a method for small molecules, especially aimed at sugar moieties, seems a good one to fill it.  So I have no objection at all to the proposed usage.'

Bea Perks

 

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References

H Tanaka, N Fukuda and Y Shoyama, J. Ag. Food Chem., DOI: 10.1021/jf063457m

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