We are developing a panel of antibodies for flow cytometry but several of the antibodies are of the IgM isotype. We are trying to find a kit or reagent to directly conjugate the antibodies to different fluorophores but most of the commercially available kits (Apex, Zenon, Lightning-Link, etc.) appear to be primarily for use with IgG isotype antibodies. Has anyone used these kits with an IgM isotype antibody? Was the labeling efficient? Are there other kits available or has anyone come across any protocols that work specifically for IgM? Thanks in advance for your suggestions!
Dear Sally,
the reagents will work also for IgM as they are not selective for IgG but are generally suited for labeling proteins. You may experience differences in the labeling efficiency and activity for different clones but this is also expected for IgG. The reason is that the reactive groups (amino acid side chains) can also be located in the antigen binding site (paratope) and labeling of those sites can inactivate the antibody (regardless of isotype). Some labels make the proteins more hydrophobic and they can become sticky and cause high background. Again, same for IgG and IgM, You need to watch out for how many labels you couple per antibody molecule. You can use the protocols provided for IgG with the exception that you can't use protein-A or protein-G affinity chromatography (some protocols may suggest that).
Cheers Markus
The company Dyomics can supply fluorescent reagents that have long Stokes shifts. These can be useful if you wish to discriminate between antibodies labelled with different fluorophores.
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