BioPath Online

Pathway Focus: Neurodegeneration


Measure neurodegenerative markers with confidence – ELISA kits for Aβ, APP, α-Synuclein and Tau | Read More
Study neurodegenerative diseases by measuring OXPHOS enzyme activity - Mitochondrial enzyme activity assays | Read More
Clever chemistry reveals an enigma of glycobiology - Click-iT® O-GlcNAc Enzymatic Labeling System | Read More


Cell-based assays for interrogating Parkinson’s disease targets - Dopamine D2 and serotonin 5-HT1A GPCRs | Read More



New Antibodies

New Immunoassays

New Molecular Probes® Products

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.
 Cerebrospinal fluid
DilutionMeaured
(pg/mL)

Expected
(pg/mL)

%
Expected

 1/2 68.99 69.99 100%
 1/4 36.90 34.50 107%
 1/8 19.43 17.25 113%
 1/16 9.09 8.62 105%

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.


Expression of α-Synuclein in various cell lines was detected by α-Synuclein ELISA.
Note consistent data of α-Synuclein measurement between ELISA (bar graph) and Western blot.


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:

  • Monoclonal capture antibodies immunocapture fully intact, active target enzymes from tissue or cell samples.
  • Enzyme activity is analyzed by measuring the change in absorbance of either the substrate or the product of the reaction (depending upon which enzyme is being analyzed).
  • By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.


Figure 1. Developed dipsticks from a 1:2 dilution series using a positive control sample and the associated standard curve. Starting material was 30 μg of fibroblast protein extract.




Product Cat. No.
 
Mitoprofile® Dipstick Kit for Complex I Activity
KHM1011
Mitoprofile® Dipstick Kit for Human Complex IV ActivityKHM1041
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 ActivityKMM2041
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:

  • The Click-iT® O-GlcNAc Enzymatic Labeling System easily attaches a click-iT® azide handle to O-GlcNAc-modified glycoproteins from pure proteins, cell lysates or protein extracts (Figure 1).
  • Detection of the labeled protein is achieved using “click” chemistry—a copper-catalyzed reaction between an azide and an alkyne.
  • Using either the TAMRA or biotin alkyne and the Click-iT® Protein Buffer Kit proteins, detection of O-GlcNAc is now a simple and robust two-step process.  

Read more about Click Chemistry



   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

Dopamine agonists are the first-line treatment for Parkinson’s disease and many act through the dopamine D2 G-protein coupled receptor (GPCR). Meanwhile, members of the serotonin 5-HT family of GPCRs, including 5-HT1A, are potential therapeutic targets of Parkinson’s disease. The most widely used treatment for Parkinson’s disease is dopamine replacement therapy. Other dopamine receptor agonists used to treat Parkinson’s include Bromocriptine, Pergolide, and Apomorphine. Interrogation of dopamine D2 and Serotonin 5-HT1A GPCRs using these three drugs with two cell-based assay technologies is discussed below.    

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