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Your Position: Casa > Fibrillas preformadas (PFF)--Un enfoque novedoso para modelar la neurodegeneración

Fibrillas preformadas (PFF)--Un enfoque novedoso para modelar la neurodegeneración

PFFs, Un enfoque novedoso para modelar la neurodegeneración
Fibrillas preformadas (PFF) Antecedentes
Schematic presentation of protein misfolding and aggregation
Schematic presentation of protein misfolding and aggregation

La agregación de proteínas es una de las principales características patológicas de las enfermedades neurodegenerativas, como la enfermedad de Alzheimer (EA), la enfermedad de Parkinson (EP), la esclerosis lateral amiotrófica (ELA) y la enfermedad de Huntington (EH). En condiciones patológicas, la Tau, la beta amiloide, la alfa-sinucleína, la TDP-43, la huntingtina y otras proteínas se integran en una amplia gama de estructuras indeseables, que van desde oligómeros y ensamblajes prefibrilares hasta agregados altamente ordenados. La estructura fibrilar representa una fase de crecimiento rápido de la agregación de proteínas, ya que estas fibrillas son "activas" y reclutan rápidamente monómeros para su elongación. Además, estas fibrillas se rompen aleatoriamente en fragmentos más cortos que podrían actuar como "semillas", que se transmiten a otras células y reclutan independientemente monómeros para formar nuevas fibrillas. Las fibrillas preformadas (PFF) son fibrillas activas formadas in vitro que tienen esta actividad de "siembra" y son capaces de reclutar continuamente proteínas patológicas endógenas solubles para formar agregados y finalmente inducir patologías neurodegenerativas.

El establecimiento de modelos de enfermedad fiables es crucial para descubrir los mecanismos patológicos, evaluar la eficacia de las intervenciones terapéuticas y valorar la seguridad de los candidatos a fármacos. En comparación con los enfoques tradicionales de modelización de enfermedades, la patología inducida por PFF no depende de la edición de genes, ni de daños químicos o físicos, y puede imitar mejor los procesos de estados patológicos que se producen de forma natural. Por lo tanto, los PFF son un enfoque novedoso para modelar las enfermedades neurodegenerativas.

Fibrillas preformadas (PFF) Producción y validación

PFFs could be generated from monomers either by incubating at 37°C and shaking, or by heparin induction. Quality control and precise initial preparation are important for successful experiments. One important aspect of successful induction of PFFs is the use of high-quality monomer with high purity, high concentration and correct conformation. Meanwhile, since the application of PFFs is cellular and animal experiments, the control of endotoxin is also an important factor. In addition, prior to the use of PFFs, PFFs should become a length of 50 nm or shorter via ultrasound to ensure the recruiting activity of PFFs as well as to facilitate the endocytosis of PFFs.

The morphology and activity of PFFs could be verified by electron microscope and thioflavin T (ThT) fluorescence assay respectively. Successfully induced PFFs show fibril structure under electron microscope. ThT assay is a classic assay to detect β-sheet structures. PFFs acquire more and more β-sheet structures as they recruit monomers, and when ThT binds to the β-sheet structure, the fluorescence value increases, thus reflecting the activity of PFFs.

Fibrillas preformadas (PFF) Producto

Aneuro es la marca de ACROBiosystems que se centra en el campo de la neurociencia. Aneuro proporciona PFF de Tau-441, PFF de alfa-sinucleína, PFF de beta amiloide, PFF de TDP-43 y PFF de SOD-1, apoyando y acelerando el establecimiento de modelos neurodegenerativos fiables.

PFFs Lista de productos

Meanwhile, alpha-synuclein PFFs,Amyloid beta PFFs,TDP-43 PFFs,SOD-1 PFFs are under development.  Deje un mensaje

PFFs Características del Producto

Producido por monómeros de alta calidad: la pureza y la homogeneidad del monómero es ≥90% según lo verificado por MALS, más propicio para la formación de PFF y asegurar la actividad.
Menor endotoxina: endotoxina monomérica ≤ 1.0 EU/μg, adecuada para diversos experimentos in vitro e in vivo.
La morfología de agregación y la actividad de reclutamiento de los PFF se verifican mediante microscopía electrónica y ensayo de fluorescencia ThT. Los protocolos son gratuitos y se comparten.
Productos y servicios personalizados con una variedad de soluciones de etiquetado fluorescente: Tinción de estrellas, etiquetado con biotina, etiquetado químico, etc.
Plazo de entrega estable, control estricto para garantizar una alta consistencia entre lotes, altamente rentable.

PFFs Datos de verificación del producto

PFFs morphology (electron microscopy)
As shown in the figure below, the PFFs have distinct fibrous structures under electron microscopy, demonstrating that our PFFs products have accurate morphology.
PFFs morphology (electron microscopy)

TEM of Human Tau-441/2N4R Pre-formed Fibrils Protein (Cat. No. TAU-H5115).

PFFs morphology (electron microscopy)

TEM of Human Tau-441 K18 (P301L) Pre-formed Fibrils Protein (Cat. No. TAU-H5113).

PFFs Bioactivity (cell base) is consistent with the reference[3]
Tau PFFs (TAU-H5115) induced significant protein aggregation in the cytoplasm of HEK293/Human Tau-K18 (GFP) Stable Cell Line (Cat. No. CHEK-ATP087).
PFFs bioactivity (cell-base assay)

HEK293/Human Tau(GFP) Stable Cell Line were transduced with Human Tau-441 / 2N4R Pre-formed Fibrils Protein, Tag Free (ThT active) (Cat. No. TAU-H5115) and Human Tau-441 / 2N4R Protein, Tag Free (MALS verified) (Cat. No. TAU-H5117) respectively. The fluorescence of GFP-Tau (Green) and DAPI (Blue) were detected by confocal microscope. A. Lipo2000 transduction. B. Lipo2000 and Human Tau-441 / 2N4R Protein, Tag Free (MALS verified) transduction. C. Lipo2000 and Human Tau-441 / 2N4R Pre-formed Fibrils Protein, Tag Free (ThT active) transduction. Scale bars, 50 μm.


PFFs activity (ThT assay)
PFFs induced faster aggregation of monomer proteins than that of the control group with only monomer added.
PFFs activity Verification (ThT assay)

Thioflavin T emission curves show increased fluorescence (correlated to tau aggregation) over time when tau wild-type monomers (Cat. No. TAU-H5117) are combined with tau wild-type Pre-formed Fibrils (Cat. No. TAU-H5115).


Modeling Neurodegenerative Diseases in Organoids with PFFs
Developed using our Cerebral Organoid Differentiation Kit (Cat. No. RIPO-BWM001K), cerebral organoids were incubated with varying concentrations of Tau PFFs to construct an AD model. Anti-pTau antibodies (Cat. No. PT1-Y2073) were used to visualize Tau aggregation alongside MAP2 staining to visualize mature neurons.
PFFs activity Verification (ThT assay)

Aggregation of Tau was visualized in organoids incubated with Tau PFFs, represented by the thickening of lines in red. Increasing concentration of PFFs resulted in a more noticeable aggregation of Tau.

Developed using our Cerebral Organoid Differentiation Kit (Cat. No. RIPO-BWM001K), cerebral organoids were incubated with varying concentrations of α-syn PFFs to construct a PD model. TH staining was used to visualize dopaminergic neurons, with MAP2 representing mature neurons.
PFFs activity Verification (ThT assay)

With the addition of α-syn PFFs into the organoid model, swelling of axon microtubules and neuron density loss is visualized. In comparison to control, significant microtubule loss (thin green lines) suggests oxidative stress stemming from addition of PFFs.

Referencias

  • 1. Stroo E, Koopman M, Nollen EA, Mata-Cabana A. Cellular Regulation of Amyloid Formation in Aging and Disease. Front Neurosci (2017). doi: 10.3389/fnins.2017.00064. PMID: 28261044; PMCID: PMC5306383.
  • 2. Guo JL, Lee VM. Seeding of normal Tau by pathological Tau conformers drives pathogenesis of Alzheimer-like tangles. J Biol Chem (2011). doi: 10.1074/jbc.M110.209296. Epub 2011 Mar 3. PMID: 21372138; PMCID: PMC3083182.

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