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In this issue:

• Gold Labeling for Cryoelectron Microscopy and Electron Tomography
  
• Dealing with Disulfides:
  Alternatives to DTT or Mercaptoethylamine Reduction

• Discussion
• References
• More information

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.

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).

• Abstract (courtesy of Nature Molecular therapy)
• Reprint (PDF) (courtesy of Nature Molecular therapy)

More information:

• Nanogold® labeling reagents – catalog and general information
• Monomaleimido Nanogold® – instructions
• Nanogold® labeling reagents – technical help FAQ and discussion
• Guide to gold cluster labeling: tips and tricks for better results
• Alternative methods for IgG labeling