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Pathway Focus: Neurodegeneration
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Measure neurodegenerative markers with confidence
| Amyloid beta (Aβ), Tau and α-Synuclein are associated with neurodegenerative diseases, most notably Alzheimer’s Disease (AD). AD is characterized by extracellular plaques and intracellular neurofibrillary tangles in the brain. Major components of the plaques are Aβ peptides, which form after sequential cleavage of amyloid precursor protein (APP). The Aβ40 form is more common, but Aβ42 is the more fibrillogenic and is thus associated with disease states. Tau’s hyperphosphorylated form is the major component of paired helical filaments, the building block of neurofibrillary lesions in AD. Hyperphosphorylation impairs microtubule binding function of tau, destabilizing microtubules in AD brains and leading to neuronal degeneration. Deposition of filamentous tau is implicated in other neurodegenerative diseases including cortical basal degeneration (CBD), progressive supranuclear palsy (PSP), Pick’s disease, and certain forms of Parkinson’s disease. α - Synuclein is highly expressed in the substantia nigra, thalamus, hippocampus, amygdala, corpus callosum, and caudate nucleus, appearing both in neurons where it concentrates at synapses, and in glial cells. α - Synuclein appears to play a role in dopamine metabolism and vesicle trafficking. Invitrogen™ sandwich ELISA kits quickly detect and quantify neurodegenerative markers in normal and diseased states. Calibrated standard curves are provided to accurately quantify the level of protein in each experimental run. The ELISA technology allows for a more detailed understanding of protein levels in neurodegenerative diseases. Please visit www.invitrogen.com/ELISA to see more ELISA kits including a selection of site-specific phosphorylated Tau ELISA kits. |
Linearity of dilution over the range of the Aß42 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.
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| Product | Cat. No. | |
| Hu Abeta 40 ELISA Kit (1 plate) | KHB3481 |  |
| Hu Abeta 40 ELISA Kit (2 plates) | KHB3482 | |
| Hu Abeta 42 ELISA Kit (1 plate) | KHB3441 | |
| Hu Abeta 42 ELISA Kit (2 plates) | KHB3442 | |
| Hu Abeta 42 High Sensitivity ELISA Kit (1 plate) | KHB3544 | |
| Hu Aggregated Abeta ELISA Kit | KHB3491 | |
| Hu APP ELISA Kit | KHB0051 | |
| Hu α-synuclein ELISA Kit | KHB0061 | |
| Hu Tau Total ELISA Kit (1 plate) | KHB0041 | |
| Hu Tau Total ELISA Kit (2 plates) | KHB0042 | |
Study neurodegenerative diseases by measuring OXPHOS enzyme activity
| Dysfunction of key mitochondrial enzyme complexes leads to not only decreased ATP production but also increased generation of reactive oxygen species (ROS). This ROS increase damages cell membranes and increases mitochondrial DNA mutation rates, leading to further enzyme dysfunction, and more cell and tissue damage. The brain’s high fatty acid content and rate of oxygen consumption makes it more vulnerable than other organs to this type of oxidative damage. For this reason, mitochondria function is implicated in many neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and ALS. Invitrogen offers unique microplate and novel dipstick enzyme activity assays to quickly assess OXPHOS enzyme complexes:
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| Product | Cat. No. | |
| Mitoprofile® Dipstick Kit for Complex I Activity | KHM1011 | |
| Mitoprofile® Dipstick Kit for Human Complex IV Activity | KHM1041 | |
| Mitoprofile® Dipstick Kit for Rodent Complex IV Activity | KRM1041 | |
| Mitoprofile® Dipstick Kit for PDH Activity | KHM1061 | |
| Mitoprofile® Human Complex IV Activity | KHM2041 | |
| Mitoprofile® Complex V Activity | KHM2051 | |
| Mitoprofile® Mouse Complex IV Activity | KMM2041 | |
| Mitoprofile® Rodent Complex IV Activity | KRM2041 | |
O-GlcNAc–clever chemistry reveals an enigma of glycobiology
| The O-GlcNAc modification is an abundant, highly dynamic, intracellular regulatory modification found in all eukaryotic cells. Like phosphorylation, O-GlcNAc modification significantly alters target protein function. In addition to its roles in signaling and protein expression, degradation, and turnover, O-GlcNAc is emerging as a key modification in the progression of several important disease states, including Alzheimer’s disease. Many tools detect and analyze proteins in depth, including such “classics” as westerns and mass spectrometry. But several important or emerging post-translational modifications (PTMs) like O-GlcNAc do not always have a suitable antibody available or are too labile, even when mass spectrometry is used, so this PTM is lost. As a result, the role of O-GlcNAc in normal and disease states remains a mystery. | Harnessing powerful click chemistry, Invitrogen offers a novel tool to detect O-GlcNAc-modified proteins:
Read more about Click Chemistry |
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Figure 1. |
| Product | Cat. No. | |
| Click-iT® O-GlcNAc Enzymatic Labeling System (for O-linked GlcNAx glycoproteins) | C33368 | |
| Click-iT® Protein Reaction Buffer Kit | C10276 | |
| Tetramethylrhodamine (TAMRA) azide (5-isomer) | T10182 | |
| Biotin azide | B10184 | |
Cell-based assays for interrogating Parkinson’s disease targets dopamine D2 and serotonin 5-HT1A GPCRs
 GeneBLAzer® D2-Gqo5-NFAT-bla CHO-K1 The GeneBLAzer® D2-Gqo5-NFAT-bla CHO-K1 cell line monitors activation of the D2 GPCR via a beta-lactamase reporter gene. The cell line contains the stable integration of human dopamine receptor 2 (D2), beta-lactamase (bla) reporter gene under control of the nuclear factor of activated T cell response element (NFAT), as well as the chimeric G-protein Gqo5 in the CHO-K1 cell line. The beta-lactamase reporter system uses a membrane-permeable FRET-based substrate, which allows measurement of activity in living cells; the dual emission wavelength read-out significantly reduces experimental variables. | Results presented here show that D2 receptor agonism can be detected for the Parkinson’s drugs bromocriptine, pergolide, and apomorphine, using the GeneBLAzer® D2 CHO-K1 cell line in a high-throughput format. GPCR-mediated signaling is part of a diverse network converging on the MAPK pathway, resulting in phosphorylation and activation of extracellular-signal regulated kinase 1/2 (ERK). Therefore, measuring ERK phosphorylation can serve as a surrogate marker for GPCR activation. We have developed a platform to monitor intracellular phospho-ERK levels using a modified baculovirus (BacMam) and a time-resolved (TR)-FRET based immunoassay. BacMam serves as the gene delivery tool that enables expression of a GFP-ERK2 sensor in mammalian cells. Subsequently, these cells expressing GFP-ERK2 are stimulated to promote ERK phosphorylation. The cells are then lysed in the presence of a Terbium-labeled anti-phospho-ERK2 antibody that binds GFP-ERK2 dually-phosphorylated at the Thr 185/Tyr 187 motif. This association allows an increased TR-FRET signal to occur between excited-state terbium and GFP, while the time-resolved dual wavelength readout significantly reduces experimental variables. Applying this platform to a CHO-K1 cell line stably expressing the serotonin 5-HT1A GPCR, we demonstrate ERK activation for the Parkinson’s drugs bromocriptine, pergolide, and apomorphine. Get more information about this novel approach by emailing us at discoverysciences@invitrogen.com |
 (click for larger view) | Measurement of dopamine D2 activation or inhibition using GeneBLAzer® technology GeneBLAzer® D2-Gqo5-NFAT-bla CHO-K1 were plated in a 384-well format. (A) Cells were activated with serial dilutions of pergolide, bromocriptine, or apomorphine and loaded with LiveBLAzer™-FRET B/G Substrate. Fluorescence emission ratios were normalized such that 0% is equivalent to the absence of stimulant while 100% equals that observed with 1 µM dopamine. (B) Inhibition of apomorphine-induced activation by perphenazine. A serial dilution of perphenazine was incubated prior to stimulation with 18 nM apomorphine. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate. Fluorescence emission ratios were normalized such that 0% is equivalent to the absence of stimulant while 100% equals that observed with 18 nM apomorphine. |
 (Click for larger view) | Measurement of 5-HT1A receptor activation using BacMam ERK technology (A) Phospho-ERK activation in BacMam GFP-ERK transduced 5-HT1A CHO-K1 cells that were stimulated with pergolide, bromocriptine, or apomorphine. Assay media was removed and 20 µL of lysis buffer with 2 nM LanthaScreen® Tb-anti-ERK2 [pThr 185/ pTyr 187] antibody was added. Phospho-ERK activation is expressed as a percentage with 100% equal to the TR-FRET ratio observed in the presence of 4 µM 5-HT and 0% equal to the TR-FRET ratio observed in the absence of ligand. (B) Inhibition of phospho-ERK activation in bacMam-GFP-ERK2 transduced 5-HT1A CHO-K1 cells by WAY-100,635. |
| Product | Cat. No. | |
| GeneBLAzer® D2-Gqo5-NFAT-bla CHO-K1 | K1708 | |
| LiveBLAzer™-FRET B/G Substrate | K1095 | |
| GFP-ERK transduced 5-HT1A CHO-K1 | K1712 | |
| LanthaScreen® Tb-anti-ERK2 [pThr 185/ pTyr 187] | PV5269 | |
