The Human c-Myc (Total) ELISA research-use-only kit is designed to detect and quantify the level of c-Myc protein independent of its phosphorylation state. This assay is intended for the detection of c-Myc in human cell lysates using 96-well plates and a microplate reader. Reactivity of this ELISA kit with other species is not assured.
Performance characteristics • Sensitivity: <5 pg/mL • Standard curve range: 15.6–1,000 pg/mL • Sample types: cell lysates • Species cross-reactivity: human • Sample volume: 50 μL (1:10) • Total assay incubation time: 4 hrs
Principle of the method The Human c-Myc (Total) kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody specific for human c-Myc (regardless of phosphorylation state) has been coated onto the wells of the microtiter strips provided. The plates are machine-coated for low well-to-well variability, and consist of removable 8-well strips, allowing you to run as few samples as you wish. The human c-Myc standard provided in the kit can be used to create a standard curve and obtain quantitative results, in addition to being used as a positive control. Samples, standard, control specimens, and unknowns, are pipetted into the wells. During the first incubation, the human c-Myc antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for human c-Myc is added to the wells.
During the second incubation, this antibody serves as a detection antibody by binding to the immobilized human c-Myc protein (regardless of phosphorylation state) captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase–labeled anti-rabbit IgG (anti-rabbit IgG-HRP) is added. This binds to the detection antibody to complete the four-member sandwich. After a third incubation and washing to remove all the excess anti-rabbit IgG-HRP, a substrate solution 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 Human c-Myc (Total) present in the original specimen.
Background information The c-Myc protein is a 57 kDa transcription factor. c-Myc activates the transcription of many target genes, including cell cycle cyclin D2 and CDK2, and translation initiation factors eIF2 and eIF4. Like other Myc transcription factors, n-Myc and L-Myc, c-Myc has a helix-loop-helix leucine zipper motif at the carboxyl-terminal domain. At the amino-terminal domain, there are two highly conserved regions required for the activation of target genes. c-Myc’s transcription activity involves complexes of several proteins. The protein MAX binds to the helix-loop-helix leucine zipper region of c-Myc. This dimerization with MAX triggers the binding of c-Myc-MAX to the DNA E-box sequence. Subsequently, c-Myc recruits histone acetyltransferase and other members of the chromatin remodeling complex. The amino-terminal domain plays an important role in the recruitment of the chromatin remodeling complex; one of the highly conserved regions in this domain binds with transformation/transcription domain-associated protein, a component of the remodeling complex.
Another binding partner for the MAX protein is MAD. MAX-MAD heterodimer binds to the DNA sequences of c-Myc’s target genes and represses the expression of these genes through the activity of histone deacetylases. Interactions between c-Myc, MAX, and MAD are critical in the control of cellular functions. When these interactions become unbalanced, such as the increased expression of c-Myc, tumors can develop. Elevated c-Myc expression is found in many cancer types.
Detect low-expressing proteins and use less sample ELISA kits designed to measure intracellular signaling targets are typically 2–10 times more sensitive than western blotting. The improved sensitivity enables you to detect low-expressing proteins that otherwise may not be distinguishable from background. In addition, the amount of sample needed to run the assay is less than what is needed for western blots.
Each Human c-Myc (Total) ELISA kit is validated for sensitivity, specificity, precision, and lot-to-lot consistency. See protocol insert for more information on validation.
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