Hey everyone, has anyone run into weird cross-reactivity problems when they're picking secondary antibodies that are supposed to be specific just for the mu heavy chain on IgM? Like, I was doing some flow cytometry work last year trying to look at early B cell responses in these mouse samples, and I used what was labeled as a super-specific anti-mu secondary. Turns out it was picking up faint signals from other stuff that shouldn't have been there—maybe leftover IgG bits or something shared across classes—and it totally threw off my gating. Spent weeks troubleshooting before realizing it wasn't my primary or the cells acting up. Super frustrating, especially when you're on a tight deadline. Anyone else dealt with this kind of unexpected binding or have tips on avoiding it? Would love to hear real experiences.
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Working Mothers
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Yeah, I've bumped into similar headaches a couple times now. Those mu-specific secondaries are great in theory because they target the unique heavy chain part of IgM, which helps dodge a lot of the usual light chain overlap you get with other isotypes. But sometimes even the cross-adsorbed ones let through low-level reactivity, especially if your sample has high endogenous immunoglobulins or if there's any species mismatch creeping in. I always double-check how the manufacturer adsorbed it—against IgG or other classes—and still, in multiplex setups, things can get messy. For anyone curious about the basics of what IgM actually involves, someone shared a decent rundown here: lgm full form. In my opinion though, it's more about testing a few different clones in your exact conditions rather than trusting the spec sheet blindly. Saved me a lot of grief after one bad batch.