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Pathway Focus: Neurodegeneration
Beta amyloid (Aβ) and tau proteins have been associated with neurodegenerative diseases—most notably Alzheimer’s disease (AD). AD is characterized by the presence of extracellular plaques and intracellular neurofibrillary tangles in the brain. The main component of the plaques is Aβ, which is formed after sequential cleavage of the amyloid precursor protein (APP). The Aβ40 form is more common, but Aβ42 is more fibrillogenic and thus is associated with disease states. Tau, in its hyperphosphorylated form, is the major component of paired helical filaments, the building blocks of neurofibrillary lesions in AD.
|Hyperphosphorylation impairs the microtubule-binding function of tau, resulting in the destabilization of microtubules in AD brains, ultimately leading to neuronal degeneration. |
ELISA technology allows a more detailed understanding of protein levels in neurodegenerative diseases such as Alzheimer’s and Parkinson’s. We offer sandwich ELISA kits that quickly detect and quantify neurodegenerative markers in human or mouse samples of cell culture supernatant, cerebral spinal fluid (CSF), and tissue homogenate for research use only. ELISA kits allow results to be collected in an easy and reproducible fashion. Calibrated standard curves are provided to accurately quantify the level of protein in each experimental run.
|Figure 1.The ELISA protocol typically requires only 4 hours of incubation time. Simply add the sample to the well, and with a few incubation steps and washes, quantitative results are available.|
|Table 1. Linearity of dilution over the range of the Aβ 42 High Sensitivity ELISA Kit. Human CSF containing 280 pg/mL of measured β-amyloid 1–42 was initially diluted 1:2, then serially diluted in standard diluent buffer over the range of the assay.|
Through autophagy, cells digest damaged or defective molecules, effectively eliminating stressors such as misfolded proteins and damaged organelles that can threaten cell health. LC3B plays a critical role in autophagy and can be used as a general marker for this vital process.
Premo™ Autophagy Sensors enable the temporal and spatial visualization of LC3B. These reagents combine the selectivity of an LC3B–fluorescent protein (LC3B-FP) chimera with the transduction efficiency of BacMam technology, facilitating unambiguous visualization of this protein with an easy one-step protocol.
|Simply add BacMam LC3B-FP reagent to cells, incubate overnight for protein expression, then image and analyze. The kits include an LC3B-FP mutant, which serves to indicate whether punctate staining may be due to FP aggregation, and chloroquine, which causes accumulation of autophagosomes.|
Using Premo™ Autophagy sensors, researchers can investigate the role autophagy plays in neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s in real time.
|Figure 2. Detecting autophagy with the Premo™ Autophagy Sensor and fluorescence microscopy. U2OS cells were cotransduced with Premo™ Autophagy Sensor LC3B-RFP and Cellular Lights™ MAP4-GFP. The following day, cells were treated with 50 µM chloroquine. The next day, cells were incubated with 1 µg/mL Hoechst 33342, then imaged using a DeltaVision® Core microscope and standard DAPI/FITC/TRITC filters.|
The innate immune response has been described as one of the factors involved in the progression of neurological disorders. Transcription factors such as IRF2 and IRF7 are important in understanding this response. Because the cause of most neurodegenerative disorders is the death of neurons and supporting cells, studying inflammation, cell proliferation, and apoptosis is essential to understanding these disorders. Targets such as Smad2, PKR, survivin, and securin are important markers for studying cell proliferation, apoptosis, and the cell cycle.
ABfinity™ antibodies are rabbit monoclonal antibodies generated by cloning specific antibody genes and expressing them in a mammalian system.
|Because gene expression is a more highly reproducible process than antibody production by hybridoma, ABfinity™ technology is designed to produce highly specific and reproducible antibodies with little-to-no lot-to-lot variance. New ABfinity™ antibodies against IRF2, IRF7, securin, survivin, PKR, and Smad2 are perfect tools for studying inflammation, cell proliferation, and apoptosis. The Smad2 [pT8] ABfinity™ Recombinant Rabbit Monoclonal Antibody is a phosphorylation site–specific antibody that detects Smad2 when phosphorylated at threonine 8.|
|Figure 3. Western blot of HeLa cell lysate treated with TGF-β using Smad2 [pT8] ABfinity™ Recombinant Rabbit Monoclonal Antibody. Rabbit anti-Smad2 pT8 (1 µg/mL) was used to label phosphorylated Smad2 in TGF-β–treated HeLa cell lysate. Phosphorylation of Smad2 at threonine 8 increased upon treatment of HeLa cells. Cells were serum starved overnight (lane 1) before the addition of TGF-β (10 ng/mL for 30 min) (lane 2). Lanes 3–5 represent competition experiments on lysate from HeLa cells treated with TGF-β as before. The antibody was used at 1 µg/mL, no peptide (lane 3), and was preincubated with the nonphosphopeptide (lane 4) or with the phosphopeptide (lane 5) to show antibody specificity to the pT8 site. Each lane was loaded with 30 µg of lysate. Western blotting was performed using the WesternBreeze® Chromogenic Kit, with NBT/BCIP as the substrate.|
|Figure 4. Immunocytochemistry of HeLa cells labeled with IRF2 ABfinity™ Recombinant Rabbit Monoclonal Antibody. HeLa cells were detected with rabbit anti-IRF2 (5 µg/mL) and labeled with Alexa Fluor® 488 goat anti–rabbit IgG at a 1:1,000 dilution. Cells were fixed using methanol fixation. The nuclear localization of IRF2 is shown in green. Nuclei were stained using SlowFade® Gold Mounting Medium with DAPI, shown in blue.|
|Description||Reactive Species||Application||Qty||Cat. No.|
|Smad2 [pT8] ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified||H (B, D, M, R, X)||WB||100 μg||700050|
|PKR ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified||H (R)||IF, WB||100 μg||700286|
|Survivin ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified||H (B, Ha, M, R)||IF, WB||100 μg||700387|
|IRF2 ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified||H (B, M, R)||IF, WB||100 μg||700226|
|Securin ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified||H (B, M)||IF, WB||100 μg||700791|
|IRF7 ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified||H||WB||100 μg||700229|
Reactivity: B = bovine; D = Drosophila; H = human; Ha = hamster; M = mouse; R = rat; R = Rhesus monkey; X = Xenopus. Applications: IF = immunofluorescence; W = western blot.
Postmortem histological examination of brains affected by Alzheimer’s disease (AD) reveals two prominent features: intracellular neurofibrillary tangles and extracellular senile plaques. The major protein components of neurofibrillary tangles are hyperphosphorylated forms of the protein tau. Increased concentrations of total tau and tau [pT181] (a site phosphorylated by GSK-3β) in cerebrospinal fluid (CSF) are observed in AD as well as in several other neurodegenerative diseases. The major protein component of extracellular plaques is β-amyloid peptide (Aβ), a 39–43 amino acid peptide cleaved from amyloid precursor protein by β-secretase (e.g., BACE) and a putative γ-secretase.
Evidence indicates that the increased release of Aβ42/Aβ43 leads to the abnormal deposition of Aβ and is associated with neurotoxicity in the brains of affected individuals.
|Interestingly, patients with AD have reduced levels of Aβ42 in CSF compared to unaffected individuals. Determining the content of total tau, phosphorylated tau, and the cleavage variants of Aβ in tissues can provide useful information about factors that influence the rate of production and/or accumulation of these analytes.|
We have released an exclusive panel of multiplex immunoassays for detecting and quantifying AD biomarkers. These assays were developed specifically for the Luminex® xMAP® platform. Analytes include Aβ40, Aβ42, total tau, tau [pT181], BDNF, GDNF, and the proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α. These immunoassays have been thoroughly validated for use with CSF and tissue culture supernatant samples. These assays are also suitable for use in brain tissue analysis.
|Figure 5. (A) Biomarkers in normal (oligoclonal IgG negative) CSF samples. Concentrations of Aβ42, tau, IL-1β, IL-6, IL-8, and TNF-α were obtained in a 6-plex assay using the Aβ Antibody Bead Kit, the Tau Antibody Bead Kit, and the Human Neuroscience Cytokine Core Panel. Aβ40 was determined in a single-analyte assay using the Aβ40 Antibody Bead Kit with the Neuroscience Buffer Reagent Kit. (B) Biomarkers in ventricular fluid from control and Alzheimer's disease patients. Results were obtained using the Neuroscience Assay Kits and the Aggregated Aβ Assay Kit. The authors wish to thank Dr. Thomas Montine, University of Washington Alzheimer's Disease Research Center (grant AG05136), for providing these samples.|
|Figure 6. Alzheimer's disease processes.|
GIBCO® recombinant neurotrophic growth factors are high-purity, high-bioactivity proteins that can be used in combination with GIBCO® specialty media products to stimulate growth of various neuronal cells for use in experimentation.
|Using GIBCO® neurotrophic growth factors with your media and cells allows for accurate and reproducible results you can rely on.|