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N A N O P R O B E S     E - N E W S

Vol. 11, No. 1          January 31, 2011


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In this issue:


PAGE 2:
Dealing with Disulfides:
Alternatives to DTT or
Mercaptoethylamine Reduction

If you are working with Monomaleimido Nanogold®, you will be aware that the thiols you wish to label are frequently found in the form of disulfides which you need to reduce before labeling.

Usually, the best way to do this is with a thiol-based reducing agent such as mercaptoethylamine hydrochloride (MEA) or dithiothreitol (DTT); however, these must be separated rigorously from the reduced antibody or protein, otherwise they will react with the Monomaleimido Nanogold®, and to do this, we recommend gel filtration liquid chromatography.

Related Nanoprobes products:

(Top) Nanogold-Fab’ vs. colloidal gold; (above) Nanogold® labeling reagents may be used to selectively label thiols and amines.

Labeling of Fab' fragments with Monomaleimido Nanogold®, showing reduction of F(ab')2 with dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA).


If you don't have access to gel filtration, can you use a reducing agent that does not react with the Monomaleimido Nanogold®?
Some recent results suggest yes – you can still obtain decent labeling using tris (carboxyethyl) phosphine (TCEP), and you don't need to separate it first. Kreppel and co-workers compared Monomaleimido labeling of thiols in virus particles in the presence and absence of DTT and TCEP. Although labeling was less in the presence of TCEP than in its absence, successful labeling and identification of the labeled protein by gel electrophoresis was carried out. Although gel filtration appears to provide better labeling, these results show that TCEP reduction can work if there is no way to separate a thiol-based reducing agent. 

 

Reference:

Kreppel, F.; Gackowski, J.; Schmidt, E., and Kochanek, S.: Combined genetic and chemical capsid modifications enable flexible and efficient de- and retargeting of adenovirus vectors. Mol. Ther., 12, 107-117 (2005).

More information:


Go to Page 1:

Gold Labeling for Cryoelectron Microscopy
and Electron Tomography

 

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