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Western blot analysis of human IgE was performed by loading 0.5ug of purified human IgE, IgM, IgA and IgG per well and 10ul of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-glycine polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blots were blocked with 5% milk in TBST for at least 1 hour at room temperature. Human IgE was detected at ~75kD using a human IgE monoclonal antibody (Product # MA5-14704) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse light chain secondary antibody at a dilution of 1:40,000 for at least 30 minutes at room temperature (left blot). To control for protein loading, a duplicate blot was probed with a mixture of goat anti-human IgG, IgM, and IgA (Product # 31128) and goat anti-human IgE (Product # A18801) antibodies at concentrations of 1ug/ml, followed by a mouse anti-goat IgG-HRP secondary antibody (Product # 31400) at a dilution of 1:8000 (right blot). Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG2b|
|Immunogen||Human Myeloma IgE|
|Storage buffer||PBS/potassium phosphate, pH 7.4, with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 6 months. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay Dependent|
|Immunoprecipitation (IP)||Assay Dependent|
|Radioimmune Assays (RIA)||Assay Dependent|
|Western Blot (WB)||1:500-1:2000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Product MA514704 is a smaller package size of MII0201 (formerly sold as a Seradyn product).
IgE is one of the five classes of immunoglobulins found in mammals. Monomers consist of two heavy (epsilon) chains with 4 Ig-like constant domains, and two light chains. IgE plays an essential role in type I hypersensitivity (allergic responses such as hay fever, asthma, hives, anaphylatic shock) by binding to Fc receptors on basophils and mast cells. Crosslinking of IgE bound to Fc receptors induces degranulation of mast cells and basophils that promote allergic manifestations. IgE is also functionally important for immunity against parasites.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.