Nanogold®-Antibody and Streptavidin Conjugates

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Contents Introduction Features of Nanogold® Conjugates Applications: In Situ Hybridization Electron Microscopy Light and Confocal Microscopy Blotting and Immunochemical Techniques Gel Stain Nanogold®-labeled bands Ordering and Price List Custom Labeling   Introduction Nanogold® is a better gold label. The 1.4 nm Nanogold® particle is a gold compound: it is not just adsorbed to proteins, like colloidal gold, but covalently reacts at specific sites under mild buffer conditions.

The Nanogold® particle is covalently and specifically linked to a hinge thiol on Fab' or IgG. The conjugate therefore has excellent stability compared to colloidal gold-antibody preparations.

It is the first probe offered as a Fab' conjugate, which is the smallest gold-antibody probe commercially available. These substantially smaller probes reach more antigens and provide better labeling.

They can be viewed directly in TEM without silver enhancement or developed with silver to any appropriate size for enhanced visibility with other counterstains.

Whereas the stoichiometry of conventional gold probes particles to IgG molecules varies from 0.2 to 10, Nanogold® preparations contain close to one Nanogold® to one Fab' or IgG.

Features of Nanogold® Conjugates

• Smallest commercially available gold immunoprobes.
• Penetrates and reaches antigens inaccessible to other probes: proven penetration up to 40 m into
• Extremely uniform 1.4 nm diameter gold label.
• Fab', IgG and Streptavidin conjugates available.
• Close to 1 Nanogold® particle to 1 Fab' (or IgG).
• Low non-specific affinity gives minimal background.
• Ultrasensitivity with silver enhancement: 0.1 pg of antigen detection on immunoblots.
• Gold is covalently attached to antibody remote from antigen binding region.
• High stability and long shelf life: conjugates show unchanged reactivity after storage for a year.
• Stable to a wide range of pH and ionic strengths.

Uses of Nanogold® Conjugates include:

• EM of ultrathin cryosections,
  see: Takizawa, T., and Robinson, J. M.; J. Histochem Cytochem., 42, 1615-1623 (1994)). [Other references].
• Pre-embedding electron microscopy. [References]
• Post-embedding electron microscopy (for methods for both pre-embedding and post-embedding,
  see: Nusser, Z., et al.: J. Neuroscience, 15, 2948-2960 (1995)). [Other references]
• Correlative electron and light/confocal microscopy (see: Sun, X.J., et al.: J. Histochem. Cytochem., 43, 329-35 (1995)).
• In situ hybridization detection. [References]
• Scanning electron microscopy (see: Hermann, R., et al.: Histochem. Cell Biol., 106, 31-39 (1996)).

Instructions and technical information (html) References for Nanogold® conjugates Technical Help for Nanogold® conjugates

Instructions (PDF)   Material Safety Data Sheet (PDF)

Applications

In Situ Hybridization

Single copies of HPV 16 in SiHa cells detected by CARD-amplified Nanogold®-silver ISH. This cell line derived from cervical carcinoma contains only one to a few copies of HPV 16, which appear as single spots. Cytospin preparation, counterstained with Nuclear Fast Red (light micrograph courtesy of Dr. G. Hacker, Landeskrankenstalten, Salzbrg. Austria).

Advantages

• More sensitive than NBT-BCIP alkaline phosphatase or peroxidase in combination with label amplification for single gene copy resolution.
• No need for more lengthy PCR procedures for most cases: avoids false positives due to mispriming and amplicon diffusion.
• Black color easily seen with standard brightfield microscope. No expensive fluorescent optics required, and no problems with autofluorescence or bleaching.
• Black signal color is compatible with full strength standard cell and nuclear stains (H & E, nuclear fast red, methyl green).
• High spatial resolution signal which may be used for EM studies as well.

Special report on In Situ hybridization with Nanogold®

Nanogold®-Streptavidin: instructions and technical information (html)
  Instructions (PDF)
  Material Safety Data Sheet (PDF)

Electron Microscopy

This scanning transmission electron microscope (STEM) image clearly shows that labeling with Monomaleimido-Nanogold® (arrows) occurs specifically at a hinge thiol site on the IgG molecule.

Nanogold® sets an unrivaled performance standard for electron microscopy. Nanogold® conjugates diffuse easily tens of microns into tissues, permeabilized cells and nuclei, where most colloidal gold probes have difficulty. Staining is more stoichiometric, with lower backgrounds. Although the 1.4 nm Nanogold® particles are visible directly at 50-120 kX magnification even in 80 nm sections (in otherwise unstained samples) they are more easily visualized after a brief (1-5 min) enhancement with silver developer (LI Silver or HQ Silver). The resulting 2 to 100 nm silver grains (size depends on development time) are easily seen at lower magnification even with uranyl acetate, osmium, or lead citrate staining.

Nanogold®-antibody conjugates	Nanogold®-streptavidin
Instructions and Product information (html)	Instructions and product information (html)
Product information (PDF)	Product information (PDF)
Material Safety Data Sheet (PDF)	Material Safety Data Sheet (PDF)

Light and Confocal Microscopy

Spindle microtubules labeled with anti-tubulin primary antibody followed by (LEFT) goat anti-mouse colloidal gold or (RIGHT) goat Fab' anti-mouse-Nanogold (Light micrograph courtesy of Dr. D. Vandré and Dr. R. Burry, Ohio State University. Original magnification = 1300x).

Nanogold® probes have excellent penetration into permeabilized cells and nuclei, similar to immunofluorescent probes and far better than colloidal gold probes. Nanogold®-Fab' probes are smaller than immunofluorescent IgG labels. There is absolutely no aggregation with Nanogold® probes, and they are very stable since the Nanogold® is covalently linked to the conjugate biomolecule. With a subsequent brief (5-15 min) silver enhancement (LI Silver) they give a signal visible even in ordinary brightfield optical microscopes (UV optics are not necessary) with a sensitivity that is usually better than fluorescence. Since the same Nanogold® probe can be used for the EM as well, preliminary staining can be followed in the light or confocal microscope while a parallel sample can be examined at the ultrastructural EM level. This flexibility is not available with fluorescent probes. Nanogold® is now available conjugated to streptavidin and anti-biotin Fab' for nucleic acid detection.

Nanogold®-antibody conjugates	Nanogold®-streptavidin
Instructions and Product information (html)	Instructions and product information (html)
Product information (PDF)	Product information (PDF)
Material Safety Data Sheet (PDF)	Material Safety Data Sheet (PDF)

Blotting and Immunochemical Techniques

Immunoblot with Nanogold®

Nanogold®-anti-mouse Fab' blotted against mouse IgG, developed with LI Silver (Nanoprobes). These ultra small gold particles nucleate silver deposition so well that unprecedented sensitivity is achieved. This immunodot blot shows 0.1 pg sensitivity (arrow).

Nanogold®-antibody conjugates	Nanogold®-streptavidin
Instructions and Product information (html)	Instructions and product information (html)
Product information (PDF)	Product information (PDF)
Material Safety Data Sheet (PDF)	Material Safety Data Sheet (PDF)

Gel Stain Nanogold®-Labeled Bands

Ask for application Note 1A "Detection of Nanogold®-labeled molecules on gels." Also see (a) Gregori, L.et al.: J. Biol. Chem., 272, 58-62 (1997), or (b) Wilkens, S., and Capaldi, R. A.: Arch. Biochem. Biophys., 229, 105-109 (1992).

Custom Labeling

Custom labeling of commercially available antibodies or your primary antibody with Nanogold® may be possible. Please Call 877-447-6266 (US toll-free number) or (631) 205-9490, use our custom synthesis quotation form, or e-mail us at tech@nanoprobes.com to discuss your project and obtain a quotation.