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Toxin-Induced Death of Neurotrophin-Sensitive Neurons

Selective destruction of neurons based on the use of targeted toxins has proven successful for several types of neurons (1). This chapter will describe the use of an immunotoxin to selectively destroy rat neurons that express the low-affinity neurotrophin receptor (p75NTR) (2). This immun ...

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Oligonucleotides as Inhibitors of Protein Synthesis

Duplex formation between an oligonucleotide and a strand of mRNA can effectively inhibit the expression of a specific gene via interfering with the cellular protein synthesis process. The antisense technology (1) is regarded as a powerful tool in molecular biology. Advances in the field h ...

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The IODO-GEN Method for Labeling and the Use of Retrograde Axonal Transport to Assay Neurotrophins

In order to look at the retrograde transport of neurotrophic factors from the target tissue to the innervating cell body, the neurotrophin needs to be labeled to a high specific activity. This chapter outlines methods used to bioassay the labeled neurotrophins and the advantages and disadva ...

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Stereological Estimation of Neuronal Number The Optical Disector/Cavalieri Combination

In recent years, we have witnessed an explosion in the number of techniques available for selectively perturbing the genes and molecules that regulate tissue growth and maintenance. From drugs and neutralizing antibodies, we have progressed to gene deletion, “dominant negative,” ant ...

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Molecular Weight Determinations Using Polyacrylamide Gel Electrophoresis with Tris-Tricine Buffers

Polyacrylamide gel electrophoresis (PAGE) in buffers containing the anionic detergent sodium dodecylsulfate (SDS) is a very powerful technique for small-scale separation of polypeptides and for assigning molecular weights to these molecules. However, the majority of syste ...

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Determination of Neuropeptides by Capillary Electrophoresis

In today’s world, we face a significant degree of miniaturization in many aspects of technology. Wonders of miniaturization are also occurring in the areas of physical and biological sciences. The development of a 21st-century technology has enabled investigations and processes to be p ...

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Characterization of Neuropeptide Processing by Fast Atom Bombardment Mass Spectrometry

Fast atom bombardment (FAB) was introduced as a new ionization technique (1) in 1981 by M. Barber and his coworkers. This was a breakthrough in the analysis of unstable and involatile compounds such as peptides, which were difficult to study by other ionization methods. FAB employs a particle beam co ...

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Analysis of Neuropeptides by Size-Exclusion HPLC Linked to Electrospray Ionization Mass Spectrometry

Atmospheric pressure ionization (API) interfaces have become powerful and popular tools for sample ionization (so-called soft ionization) and are primarily used in the analysis of polar and thermolabile compounds (e.g., peptides, proteins). Detailed descriptions of the most imp ...

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Identification of Peptides by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and Direct Analysis of the L

This method describes how, by mixing peptides with a UV-absorbing matrix, their masses can be determined using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

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Use of Circular Dichroism to Determine Secondary Structure of Neuropeptides

Circular dichroism (CD) spectroscopy is currently the method of choice to study the conformation of proteins in low resolution (1). Chiral substances absorb right and left circularly polarized lights to different extents, demonstrating differences in absorbance (△A) and molar exti ...

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1H Nuclear Magnetic Resonance (NMR) in the Elucidation of Peptide Structure

1H nuclear magnetic resonance (NMR) has proven to be a uniquely powerful tool for studying the structure of peptides in solution. I will concentrate on the type of structural information that is obtainable from NMR, the types of spectra needed to get this information, and how to interpret these spec ...

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The Study of Membrane- or Receptor-Bound Neuropeptides by NMR

An X-ray crystal structure of a neuropeptide bound to its native receptor protein would provide a wealth of information concerning the structural requirements for ligand-receptor binding. Unfortunately, many neuropeptide-protein complexes are very difficult, if not impossi ...

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Molecular Modeling of Neuropeptides

Molecular modeling is the science of the generation, manipulation, and representation of three-dimensional structures of molecules using computational chemistry and high resolution computer graphics. Since peptides of biological interest are large molecules, molecul ...

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Preparation of Neuropeptide-Containing Fractions from Biological Materials

Neuropeptides vary appreciably in terms of then molecular mass, charge, and hydrophobicity so that there is no single optimum method for their extraction from biological materials such as tissues, cultured neurons, plasma, or cerebrospinal fluid (CSF). As all neuropeptides are rapid ...

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Production of Antisera Using Peptide Conjugates

Since an immunogen requires both an antigenic site and a T-cell receptor binding site, there is a minimum size necessary (1). Natural immunogens have a molecular weight 5000. Small molecules such as neuropeptides may be able to bind to the surface of B-cells, but do not stimulate an immune response. Such ...

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Radioimmunoassay

There is frequently a need to measure concentrations of neuropeptides in tissue perfusates, tissue extracts, chromatographic column fractions, and so on. Since the concentrations of neuropeptides encountered are often low (usually the low fmol/mL range in perfusates and pmol/g ran ...

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Sample Preparation for Peptide Immunocytochemistry

Immunocytochemistry is the localization of a tissue constituent in situ by means of a specific antigen-antibody reaction tagged by a visible label (1). For many years, the technique was not considered sufficiently reliable for the de nova identification of substances, but was used to assess ...

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Enzyme-Linked Immunosorbent Assay of Peptides

Enzyme-linked immunosorbent assay (ELISA) is a member of the solid-phase immunoassay family that detects specific antigen-antibody binding reactions (1,2). A great many variables of antigen or antibody presentation, treatment, and detection are considered when performing EL ...

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lmmunocytochemical Methods for Regulatory Peptides

Immunocytochemistry employs antibodies to detect peptides or proteins in tissue preparations (1). However, this in itself is not a visible reaction. Several methods, employing secondary antibodies labeled in some way to render them visible, generally with fluorescent compounds or ...

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Ultrastructural Localization of Peptides Using Immunogold Labeling

The subcellular localization of bioactive peptides, including hormones and neurotransmitters, has immense value, not only in understanding how cells function, but in correlating biochemical and clinical data from tissues in both normal and diseased states. The pioneering immu ...

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