Glutathione Beads: Biochem's Little Magic Worker

Glutathione beads are necessary for a few protein isolations and deserve some amount of discussion about their use and effectivity. Science today is expensive; it's important to know your laboratory equipment.

First off, laboratory precision is absolute necessity; a difficult problem facing effective proteomic isolations is the yield of correct proteins after an elution scheme.

It is a fine coincidence of solutions, column conditions, timing, and equipment that lead to accurate laboratory results. Trouble-shooting these results is a must, and it is not a simple procedure. Furthermore, each procedure must be tailors to the empirical best yield for each protein of interest.

It's a dance of calibration, and the difficulties in finding the BEST empirical way to perform the isolation are numerous.

For instance, if there is a low protein yield, it is possible that the fusion protein does not bind to the immobilized glutathione. In this case, the conformation of the fusion complimentary protein has altered its affinity with its partner protein.

If there's too much protein, then the fusion protein has interacted with other proteins in solution, or an un-targeted peptide is stuck on the beads. (Major nightmare! Globs of eluted protein, not good! This is a waste of hours of work!)

I have read several postings of grad-students trying to find a way past odd proteins that become snagged by their Glutathione bead traps.

If there is one issue to confront with peptide isolation, it's laboratory technique. Some PhD candidate have to bank their entire scientific careers on one paper. That equates to a risk of hundreds of thousands of dollars in student loans spent hoping that one laboratory technique will work right. Maybe that's a bit extreme, but the denials for PhD candidacy are very harsh. And, the average income levels of PhDs versus Masters-level job-applicants are in the tens of thousands of dollars.

Tiny bead, big importance.

Bio-chem supply companies even produce 'designer' GSH-agarose proteins and assay kits to make specific isolations easier, for example, the GLUTATHIONE SEPHAROSE(R) 4B has a 10-carbon spacer arm for better GST binding.

Also, the beads themselves are expensive. Sloppy lab work costs money.

If there was any suggestion I would make, it's keep trying combinations until you find something that works. And consult your peers. And consult the world-wide web.

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Some general background about Glutathione:

Glutathione (IUPAC name 2-amino-5-{[2-[(carboxymethyl)amino]-1-(mercaptometh yl)-2-oxoethyl]amino}-5-oxopentanoic acid) is a tripeptide with a cysteine group and a carboxyl-glutamate side chain.

Glutathione is an excellent non-enzymatic reduction agent for (thiol) disulfide bonds, and keeps cysteine thiol side chains in a reduced state on the surface of proteins. This property also lends to glutathione's role as an anti-oxidant to trap free radicals.

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Some companies and links to Glutathione-agarose beads:

Glutathione Sepharopore 4B
http://www.bio-world.com/MoreInfo.asp?ItemCode=506 407

BD Pharmingen Glutathione Agarose Beads
http://www.bdbiosciences.com/ptProduct.jsp?prodId= 60292

Sigma-Aldrich, pre-packaged columns
http://www.sigmaaldrich.com/catalog/search/Product Detail/SIGMA/G3907

Amersham's Website
http://www4.gelifesciences.com/aptrix/upp01077.nsf /Content/Products?OpenDocument&parentid=25190034&mod uleid=166324