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SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

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Pertenece a: PubMed Central (PMC3 - NLM DTD)  

Descripción: The anomalous folding and polymerization of the β-amyloid (Aβ) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. Aβ is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into β-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Aβ multimers are more toxic than fibrillar Aβ amyloid2. The identification and quantification of low- and high-molecular weight multimeric Aβ species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring Aβ multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Aβ-specific antibodies. However, the separation and detection of multimeric Aβ requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Aβ peptide. Using this protocol, we consistently detect Aβ monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

Autor(es): Rosen, Rebecca F. -  Tomidokoro, Yasushi -  Ghiso, Jorge A. -  Walker, Lary C. - 

Id.: 55213330

Idioma: English  - 

Versión: 1.0

Estado: Final

Palabras claveJoVE Neuroscience - 

Tipo de recurso: Text  - 

Tipo de Interactividad: Expositivo

Nivel de Interactividad: muy bajo

Audiencia: Estudiante  -  Profesor  -  Autor  - 

Estructura: Atomic

Coste: no

Copyright: sí

Requerimientos técnicos:  Browser: Any - 

Fecha de contribución: 24-abr-2012

Contacto:

Localización:


Otros recursos del mismo autor(es)

  1. Mitochondrial dysfunction induced by a post-translationally modified amyloid linked to a familial mutation in an alternative model of neurodegeneration Familial British dementia (FBD) is an early-onset non-amyloid-β (Aβ) cerebral amyloidosis that prese...
  2. Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

    Abstract

    Background

    Alzheimer's disease (AD) is a devastating neurodegenerative dis...


  3. Aβ seeds resist inactivation by formaldehyde Cerebral β-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts c...
  4. Context Dependence of Protein Misfolding and Structural Strains in Neurodegenerative Diseases Vast arrays of structural forms are accessible to simple amyloid peptides and environmental conditio...
  5. Clusterin and Complement Activation in Exfoliation Glaucoma This study provides experimental evidence for the involvement of the multifunctional extracellular c...

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