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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 # MA5-14712) 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 MA514712 is a smaller package size of MII0402 (formerly sold as a Seradyn product).
Immunoglobulin M (IgM) is produced as a 900 kDa pentamer, which is an efficient complement binder. This antibody type is produced initially in the immune response and it is the first immunoglobulin class to be synthesized by a fetus or newborn. IgM antibodies do not cross the placenta. IgM concentration in blood is 0.12 g/l and its biological survival (plasma T1/2) is 5 days.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.