Nanogold-Peptides (continued)

Non-Radioactive Localization of Substance P Binding Sites in Rat Brain and Spinal Cord Using Peptides Labeled with 1.4 nm Gold Particles

Abstract:

NHS-Nanogold® was used to label the bioactive peptide, Substance P (SP), which has only 11 amino acids and a moleculear weight of 1,517 daltons.

Careful controls showed that the Nanogold-SP behaved similarly to the commonly used Iodine-125-labeled SP. Specific binding to target proteins on SDS-PAGE blots could be competed off with excess unlabeled SP; binding was also inhibited by a specfic NK1-receptor antagonist, but not by neurokinin A, which does not target the NK1 receptor. Nanogold alone led to no specific staining.

The Nanogold-SP gave rapid, high resolution data mapping the receptor distribution in tissue sections of brain and spinal cord.

The authors comment: "The new method which we have developed usng Nanogold-labeled SP combines the advantages of specific receptor-ligand binding with a non-radioactive detection system. The distribution of the gold label can be vsualizedby a silver enhancement reaction without an additional immunohistochemical incubation.

"It should be possible to label a great variety of peptides that have an accessible primary amino group. in addition to SP, we have been able to label somatostatin and bradykinin successfully, and they have been used for demonstrating their binding sites in Western Blots, histological sections, and cell cultures. Other advantages are short processing time and high spatial resolution. The specificity was clearly shown by the various control experiments.

"In summary, this study shows that SP can be labeled by NHS-Nanogold to demonstrate its binding sites in histological sections. It may be possible to perform this kind of labeling on other peptides. The combination of receptor-ligand affinity binding with a non-radioactive detection system shows the following advantages:

1. a very short procedure time
2. no need for an aditional immunohistochemical step
3. no fading of the signal
4. high spatial resolution

SDS PAGE of Proteins of the rat spinal cord. lanes: (a) india ink dye stain showing many proteins (b) Iodine-125-SP stain, showing binding to 58 and 38 kD protein bands. (c) Iodine-125-SP and excess SP, showing SP competes for labeled protein binding. (d) SP-Nanogold (silver enhanced) showing binding to 58 and 38 kD bands, similar to Iodine-125-SP (lane b). (e) SP-Nanogold and excess SP, showing that excess SP competes off labeled protein (similar to lane c).

Courtesey of G. Segond von Banchet and B. Heppelmann, Physiologisches Institut der Universität Würzburg, Würzburg, Germany.

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Conformational Constraints in Protein Degradation by the 20S Proteasome

Abstract:

The authors used Monomaleimido-Nanogold® to label a thiol group on the B chain of insulin, a 30 amino acid peptide.

This Nanogold-B chain was found to insert into the proteasome, with the gold at the outer orifice, and the B chain was not degraded. The Nanogold-B chain also blocked degradation of additionally added unlabeled B chain. However, Nanogold-B chain was degraded by other proteinases (trypsin, chymotrypsin), ruling out the possibility that a tight physical interaction between the peptide chain and the gold prevented proteolytic degradation.

Nanogold was visualized directly (without silver enhancement) by high-resolution electron microscopy and negative staining in ammonium molybdate/glucose. Image processing was used to analyze the micrographs.

This data helps to understand how proteasomes function: it appears that a narrow opening controls access to the inner proteolytic compartment of the barrel-shaped proteasome.

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Localization of Calmodulin Binding Sites on the Ryanodine Receptor from Skeletal Muscle by Electron Microscopy

Abstract:

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of the skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by cryo-electron microscopy using Nanogold-labeled calmodulin.

The calmodulin from wheat germ is unusual, in that it contans a cysteine residue (Cys-27), which can be specifically linked to sulfhydryl-specific reagents. Monomaleimido-Nanogold® was used to attach the 1.4 nm Nanogold. CaM has a molecular weight of 17 kDa. SDS-PAGE was used to demonstrate biochemically the targeting of the Nanogold-CaM, and blot transfers were made and stained with silver to reveal gold-containing bands.

The authors point out: "[Nanogold] has significant advantages over the more commonly used colloidal gold probes. It can be derivatized with protein reactive groups, such as the maleimido moiety used n this study, and therefore can be directly linked to a macromolecular substrate in a specific manner. This chemical specificity, together with the small size of the [Au(1.4nm)] cluster, means thatt the clusters observed in the micrographs of macromolecules labeled with this compound should lie within 2 nm of their respective reactive sulfhydryls. In contrast, colloidal gold probes are usually attached tothe macromolecule of interest via an intermediary protein, such as a Fab fragment or streptavidin, which reduces the accuracy of localization achievable to ~8 nm or more. " Another superior property of [Au1.4nm] probes is the reported low affinity of the [Au1.4nm] itself for proteins (Hainfeld, J. F., and Furuya, F. R.; J. Histochem. Cytochem., 40, 177 (1992)), which was confirmed in this study. In contrast, the streptavidin-colloidal gold probe that we used to detect biotin-CaM bound to RyRs in electron micrographs showed levels of nonspecific binding many times higher than was found for [Au1.4nm]-CaM and, consequently, it was difficult to distinguish confidently between specific and non-specific gold-RyRs complexes."

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Ordering Information:

1.4 nm gold particle with a single N-hydroxy-sulfosuccinimide group. Used for labeling primary amines.
Instructions and technical information            Instructions in PDF Format

2025A Mono-Sulfo-NHS-Nanogold® in 5 aliquots (NHSNA)
30 nmol ($381)

Same total quantity as 2025, but provided in 5 separate tubes. Since the NHS-ester hydrolyzes once dissolved, separate aliquots are preferable if several smaller quantity labeling reactions are planned at different times.
Instructions and technical information            Instructions in PDF Format

2025S Mono-Sulfo-NHS-Nanogold® introductory size (NHSNS)
6 nmol ($90)

Try Mono-Sulfo-NHS-Nanogold® on a smaller scale for a low price.
Instructions and technical information            Instructions in PDF Format

2020 Monomaleimido Nanogold® (MMN)
30 nmol ($330)

1.4 nm gold particle with single maleimide group for selectively labeling thiols (-SH). May be used to label primary Fab' antibody fragments, IgG, cysteine groups on proteins, or other sulfhydryl containing compounds. 30 nmol provided: enough to label 0.2 mg of Fab'.
Instructions and technical information            Instructions in PDF Format

2020A Monomaleimido Nanogold® in 5 aliquots (MMNA)
30 nmol ($381)

Same total quantity as 2020, but provided in 5 separate tubes. Since the malieide moiety is unstable once dissolved, separate aliquots are preferable if several smaller-quantity labeling reactions are planned at different times.
Instructions and technical information            Instructions in PDF Format

2020S Monomaleimido Nanogold® introductory size (MMNS)
6 nmol ($90)

Try Monomaleimido Nanogold® on a smaller scale for a low price.
Instructions and technical information            Instructions in PDF Format

2021 Monoamino Nanogold® (MN)
30 nmol ($228)

1.4 nm gold particle with a single primary amine may be used for labeling the carbohydrate moiety of glycoproteins or other applications.
Instructions and technical information            Instructions in PDF Format

2021A Monoamino Nanogold® in 5 aliquots (MNA)
30 nmol ($254)

Same total quantity as 2021, but provided in 5 separate tubes.
Instructions and technical information            Instructions in PDF Format

2021S Monoamino Nanogold® introductory size (MNS)
6 nmol ($65)

Try Monoamino Nanogold® on a smaller scale for a low price.
Instructions and technical information            Instructions in PDF Format

2022 Positively Charged Nanogold® (PN)
30 nmol ($228)

1.4 nm gold particles with a net positive charge; contains multiple amines. Use these particles for alternative coupling schemes, or to bind to negatively charged sites.
Instructions and technical information            Instructions in PDF Format

2023 Negatively Charged Nanogold® (NN)
30 nmol ($228)

1.4 nm gold particles with a net negative charge; contains multiple carboxyl groups. Use these particles for alternative coupling schemes, or to bind to positively charged sites.
Instructions and technical information            Instructions in PDF Format

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Gold Labeling Reagents: Complete Catalog Information
Catalog Index

Other Applications:

Product Applications Special report: A very reliable pre-embedding immunogold method. Our 2002 paper: Nickel-NTA-Nanogold Binds his-Tagged Proteins. Our 2001 paper on DNA Nanowires. Our 1999 paper on Gold-Based Autometallography. Special report: In Situ Hybridization with Nanogold®-Streptavidin. Technical note: Nanogold® Staining on Gels. Our 1996 paper on Gold Liposomes. Special report: Nanogold® Labeling of Peptides. Technical note: RNA Labeling with Nanogold®. Our 1991 paper on A New 1.4 nm Gold-Fab' Probe. Our 1994 paper on Methylamine Vanadate (NanoVan) Negative Stain. Our 1990 paper on Conducting Polymer Films as EM Substrates. Research Applications Our 2005 paper: 5 nm Gold-Ni-NTA binds His Tags. Enzymatic Metallography: A Simple New Staining Method Our 2006 paper on Light and Electron Microscopy of Microsporida using Enzyme Metallography. Our 2004 paper on Enzymatic Metallography as a Correlative Light and Electron Microscopic Method. Our 2002 paper on Enzymatic Metallography: A Simple New Staining Method.

Research Applications (continued) Our 2007 paper on Correlative Enzymatic and Gold Probes for Light and Electron Microscopy. Our 1995 paper on Aldehyde Gold Clusters for Molecular Labeling. Our 2000 paper on Gold Cluster Crystals. Larger covalent gold probes: Our 2005 paper on Preparation of Covalent 3nm Gold – Fab’ Probes. Our 1999 paper on A Covalently Linked 10 nm Gold Immunoprobe. Combined fluorescent and gold probes: Our 2003 paper featuring Dextran-Texas Red®-Nanogold® Fluorescent Dyes for Use in Correlative Microscopy and Intravital Imaging. Our 2002 paper on Combined ALEXA-488 and Nanogold Antibody Probes. Our 1999 paper on Combined Cy3 / Nanogold Probes for Immunocytochemistry and In Situ Hybridization. Our 1998 paper on Dual-Labeled Probes for Fluorescence and Electron Microscopy. News: our original 1997 paper on Combined Fluorescent and Gold Immunoprobes. Our 1996 paper on Large Cluster and Combined Fluorescent and Gold Immunoprobes. Our 1994 paper on Combined Fluorescent and Gold Nucleic Acid Probes. Our 2005 paper on High Z Metal Carbonyls for Imaging and Microspectroscopy. Our 2005 paper on In Vivo Vascular Casting.