Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
4 hours 40 mins (after plate preparation)
Sample Type & Volume Required
Cell lysates (100 µL)
Range
312.00 - 20,000 pg/mL
Sufficient Materials
Kits available for two, five, or fifteen 96-well plates*
Specificity
Please see the
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
Development protocols are provided to guide further assay optimization
Assay can be customized to your specific needs
Available in 2, 5, and 15- (96-well) plate pack sizes
Economical alternative to Western blot
Kit Content
Capture Antibody
Conjugated Detection Antibody
Calibrated Immunoassay Standard or Control
Streptavidin-HRP
Other Reagents Required
PBS: (Catalog # ), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2HPO 4, 1.5 mM KH 2O 4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # ), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
Substrate Solution: 1:1 mixture of Color Reagent A (H 2O 2) and Color Reagent B (Tetramethylbenzidine) (Catalog # )
Stop Solution: 2 N H 2SO 4 (Catalog # )
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # ), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # ), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Data Examples
Figure 1: Specificity of Human Total MDM2 Duoset IC ELISA is shown by Western blot. Lysates prepared from LLnV treated U2OS human osteosarcoma cells were incubated in wells coated with Human Total MDM2 Capture Antibody. Unbound material was removed by washing and bound material was solubilized in SDS gel sample buffer. The same lysate and captured proteins were electrophoresed, transferred to a PVDF membrane, and immunoblotted with Human Total MDM2 Detection Antibody. The captured protein corresponds to MDM2 detected in the total cell lysates. |
Figure 2: Amounts of MDM2, as quantified by the Human Total MDM2 Duoset IC ELISA, are consistent with the relative levels of MDM2 determined by qualitative Western blot analysis. Lysates prepared from U2OS human osteosarcoma +/- LLnV cells were quantified with this DuoSet® IC. The same lysates were also immunoblotted (inset) with a monoclonal detection antibody. The DuoSet® IC results correlate well with the relative amounts of human MDM2 detected by Western Blot. |
Preparation and Storage
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: MDM2/HDM2
MDM2 is a key regulator of p53 tumor suppressor protein activity and stability. MDM2 binds to and inhibits the transactivation domain of p53. In addition, MDM2 controls p53 stability by functioning as its E3 ligase in ubiquitination and by shuttling p53 from the nucleus to the cytoplasm for subsequent degradation. The importance of the p53/MDM2 relationship is underscored by the existence of an autoregulatory feedback loop whereby activated p53 transcriptionally upregulates the expression of its own inhibitor, MDM2.
Entrez Gene IDs:
4193 (Human); 17246 (Mouse);
Long Name:
Double Minute 2 Protein
Aliases:
ACTFS; HDM2; HDMX; MDM2 oncogene, E3 ubiquitin protein ligase; MDM2
