PA5-33229 can be used as a negative control in flow cytometry when using mouse monoclonal antibodies in human cell surface antigens of isotype IgM. This enables an estimation of non-specific binding of mouse monoclonal antibodies to cell surface components. This antibody recognizes a rat cell surface marker and therefore cannot be used as a negative control in the species.
For FACS analysis, use 10 µL of the suggested working dilution to label 1x10^6 cells in 100 µL.
IgM (Immunoglobulin M) is expressed intracellularly during the early stages of B lymphopoiesis, and then on the surface of more mature B cells in the bone marrow and peripheral B cells. The isotype of a primary antibody and its application can result in background staining. Primary antibody background noise can be caused by binding to Fc receptors on target cells; by non-specific interactions with cellular proteins, carbohydrates, and lipids; or by cell autofluorescence. Isotype control antibodies can act as negative controls to help differentiate non-specific background signal from specific antibody signal because they have no relevant specificity to a target antigen. While isotype controls are most commonly used in flow cytometry, they are also useful in other applications such as chromatin immunoprecipitation (ChIP), immunohistochemistry, and gel shifts. Isotype controls should match with the primary antibody species and isotype so that the level of specific staining by the primary antibody may be accurately determined. If using directly labeled primary antibodies, the isotype control works best if conjugated with the same label as the test antibody.