• Gel Staining with Silver Enhancer: LI Silver
• Specific: Develops only Nanogold®-labeled band
• Rapid: 1-5 minutes
• Sensitive: More sensitive than usual gel stains
• May be used directly on gels or on blot transfers

1.4 nm Nanogold® particles may be developed with silver so that they become visible to the naked eye, thus amplifying the signal thousands of times. If you have used monomaleimido-Nanogold, mono-NHS-Nanogold® or mono amino-Nanogold® to label a protein or other molecule, these may then easily be analyzed and detected on gels using silver enhancement. Following is a protocol for detecting Nanogold®-labeled bands on gels or blots:

1. After labeling with Nanogold®, remove unbound gold particles by column chromatography, sucrose gradient or other purification means. Leaving excess free Nanogold® in the sample will interfere with the intended gel staining.
2. Run gel as usual with two precautions:

   A) Nanogold® is degraded by beta-mercaptoethanol (or DTT), so the sample must not be mixed with a reducing agent, i.e., a non-reducing gel must be run. Normal concentrations of other ingredients (SDS, etc.) are acceptable.

   B) Sample must not be heated. Frequently samples are boiled in SDS before loading on the gel. Since Nanogold® is degraded above 50°C, no heating is recommended. This is usually not a limitation since normal gel patterns are obtained with most samples without heating.

3. Gel may be electrotransferred to nitrocellulose if desired, although this is not necessary.
4. Rinse gel with several changes of deionized water. Since the silver developer is precipitated by halides, traces of NaCl must be removed.
5. Place the gel or blot in a suitable dish and apply enough freshly prepared LI Silver (catalog# 2013) to cover the gel. LI Silver is prepared by mixing equal amounts of A and B components. Do not use the usual gel silver stains which are quite different from LI Silver and do not develop the Nanogold® effectively.
6. Watch development of band(s) which should appear brown-black. Aggregates with gold that did not enter the gel or small amounts of free gold may give background staining. Usual development time is 1-5 min. Extensive development time (> 30 min) will lead to some non-specific background self-nucleation staining by the developer alone.
7. When optimal staining is reached, stop development by rinsing in deionized water. The final stained gel is now a permanent record.
8. For comparison and visualization of all bands, run a duplicate gel and stain with Coomassie Blue or gel silver stain.
9. A Nanogold®-labeled molecule typically runs ~15,000 MW higher on the gel due to the added weight of the Nanogold® particle (~15,000). Thus labeled and unlabeled molecules are separated, and their proportion may be estimated by usual gel stains (Coomassie or gel silver stains) which show total protein content.

References:

1. Gregori, L., J. F. Hainfeld, M. N. Simon, and D. Goldgaber. 1997. Binding of amyloid beta protein to the 20S proteasome. J Biol Chem 272 (1): 58–62
2. Wilkens, S. and Capaldi, R.A. Monomaleimidogold labeling of the gamma subunit of the Escherichia coli F1 ATPase examined by cryoelectron microscopy. Arch Biochem. Biophys., 229, 105-109 (1992).