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||Goat / IgG|
|Storage buffer||PBS, pH 7.2|
|Contains||0.05% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Cross Adsorption||Against bovine, mouse and rabbit serum proteins|
|Antibody Form||Whole Antibody|
|Tested Applications||Dilution *|
|Western Blot (WB)||1 µg/ml|
* 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.
Anti-Human secondary antibodies are affinity-purified antibodies with well-characterized specificity for human immunoglobulins and are useful in the detection, sorting or purification of its specified target. Secondary antibodies offer increased versatility enabling users to use many detection systems (e.g. HRP, AP, fluorescence). They can also provide greater sensitivity through signal amplification as multiple secondary antibodies can bind to a single primary antibody. Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species and can be further purified and modified (i.e. immunoaffinity chromatography, antibody fragmentation, label conjugation, etc.) to generate highly specific reagents.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.