The Human CREB (Total) ELISA Kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to detect and quantify the level of CREB (Total) in fresh or frozen human cell lysates. Cross-reactivity has been observed in mouse cells. The assay recognizes both natural and recombinant CREB (Total), independent of its phosphorylation state.
Principle of the method
A monoclonal capture antibody specific for CREB (Total) has been coated onto the wells of the 96-well plate. During the first incubation, standards of known content and unknown samples are pipetted into the wells and the antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for the target protein is added to the wells and serves as a detection antibody by binding to the immobilized protein captured during the first incubation. After washing, a horseradish peroxidase labeled anti-rabbit IgG is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution (TMB) is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of target protein present in the original specimen and the optical density can be read on a standard microplate reader.
Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.
CREB (Cyclic AMP response element binding protein) is a 43 kDa basic/leucine zipper transcription factor that binds the cyclic AMP response element (CRE) and activates transcription in response to a variety of extracellular signals including neurotransmitters, hormones, membrane depolarization, and growth and neurotrophic factors. Activation of CREB is dependent upon the phosphorylation of serine 133. Phosphorylation of CREB occurs via p44/42 MAP kinase and p90RSK, and via p38 MAP kinase and MSK1. Although CREB will bind DNA independent of its phosphorylation state, only the phosphorylated form is competent as a transcription factor. CREB binding protein (CBP), a transcriptional coactivator that directly interacts with CREB, binds to CREB in the region of serine 133. CREB is involved in different cellular processes including the synchronization of circadian rhythmicity, differentiation of adipose cells, and learning and memory. In humans, the gene is located on the q arm of chromosome 2. Diseases associated with CREB dysfunction include Alzheimer’s Disease, histiocytoma and soft tissue melanoma.
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