Brad Larson1, Arturo Gonzalez-Moya2, Alexandra Wolff3, Wini Luty3
1BioTek Instruments, Inc. Winooski, VT USA
2Cisbio Bedford, MA USA
3Enzo Life Sciences Farmingdale, NY USA
Featured Product: β-Amyloid (1-42),
Amyloid β A4 (CT, 1-42) monoclonal antibody (8G7),
Goat anti-mouse IgG1 (human & mouse Ig adsorbed), (ATTO 590 conjugate)
> Download the complete application note
INTRODUCTION
According to literature references, Extracellular-Signal Regulated Kinase (ERK) signaling has been linked to memory and regulated by environmental stimuli. In Alzheimer’s disease, progressive cognitive impairment is seen as a classic characteristic. These deficits are believed to be the result of progressive synaptic dysfunction initiated by aggregated β–amyloid peptide 1-42 (Aβ). Zhu
et al. (2002) showed that Aβ induced disruption of kinases critical for memory. In late stages of Alzheimer’s disease, ERK activation is suppressed relative to early stages and controls (Webster
et al. 2006).
In vitro studies have also shown that under certain conditions Aβ or fragments inhibit ERK or the downstream cAMP response elementbinding protein (CREB) in neuronal cell models (Daniels
et al. 2001).
Here we evaluate the ability to detect changes in phosphorylation levels of ERK and CREB following treatment with Aβ using the SH-SY5Y neuroblastoma cell line. Aβ was oligomerized using a method previously described in the literature (FA
et al. 2010). A two-step HTRF® assay process was incorporated such that cell plating and treatment are carried out in a 96-well clear-bottom imaging plate. Following lysis, aliquots were transferred to separate LV384-well assay plates to perform the phospho- and total ERK and CREB assays. A neutralizing antibody was also tested for its capacity to counteract the inhibitory effects of Aβ. Aβ binding and antibody neutralization were detected via
immunocytochemistry and microscopic analysis. All microplate reading and cellular imaging steps were performed using a novel cell imaging multi-mode reader. The combination provides an efficient, robust method for testing of new molecules to combat the degenerative effects of the disease.
Read More about Neuroscience
TechNotes
Our bi-weekly TechNotes are an easy way for you to keep in touch with how Enzo Life Sciences is living up to our motto: Scientists Enabling Healthcare™. Each TechNote connects recently published research with one or more of the many solutions Enzo provides for scientists across the life sciences, drug development, and clinical research communities.
Neuroscience Product Range
Fueling Discovery in the Etiology, Pathology, and Treatment of Neurodegenerative Disease.