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Western blot analysis of IgM was performed by loading 1ug of purified Human IgM, Human IgA, and Human IgG and 10ul of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% BSA in TBST for at least 1 hour at room temperature. IgM heavy chain was detected using a mouse anti-human IgM monoclonal antibody (Product # MA5-14729) diluted to a concentration of 2ug/ml in blocking buffer, overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse light chain secondary antibody diluted 1:40,000 for at least 30 minutes at room temperature (panel A). To verify the presence of all isotypes, a second blot was probed with a goat anti-human IgM + IgG +IgA polyclonal antibody (Product # 31128) at a concentration of 0.5ug/ml in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated mouse anti-goat IgG secondary antibody (Product # 31400) diluted 1:40,000 in blocking buffer for at least 30 minutes at room temperature (panel B). Chemiluminescent detection was performed using SuperSignal West Pico.
|Tested species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||Purified Human Serum IgM|
|Storage buffer||0.01M potassium phosphate with 2.5mM KCl, 0.135M NaCl, 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)||1 µg/ml|
|Flow Cytometry (Flow)||1 µg/ml|
|Immunoprecipitation (IP)||Assay Dependent|
|Radioimmune Assays (RIA)||Assay Dependent|
|Western Blot (WB)||1:100-1:400|
* 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.
By ELISA and WB, MA5-14712 does not react with Human IgG or Human IgA.
Product MA514729 is a smaller package size of MII0401 (formerly sold as a Seradyn product).
Thermo Scientific 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.