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I'm beginning to do some cell-binding assays and I would like for my proteins to not be endocytosed by my mammalian cells. Typical suggestions are for the cells to be kept on ice and that the binding experiment also occurs when the cells are chilled.

However, to be sure it has been suggested that I use endocytosis inhbitors. So my questions are, when are the endocytosis inhibitors necessary? How would I know that endocytosis inhibitors are necessary? Which ones would I actually use?

I recognize that there are both clathrin and non-clathrin mechanisms of internalization. Which mechanism would be more useful to study for an antibody-receptor interaction?

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As with so many things, it depends. Also, I'm a bit unclear when you say antibody-receptor interaction, do you mean that you have an antibody that's specific for a receptor, or that you're studying Fc receptors binding to antibodies? (for the following, I assume the former)

For most cell types and most cell surface molecules, simple antibody binding isn't going to induce endocytosis, and keeping the cells on ice is sufficient to prevent most background endocytosis (i'd do the assays in a cold room as well so that even when you take tubes out of the ice bucket they stay cold).

On the other hand, if you're using highly phagocytic cells (like macrophages), and/or you're triggering a receptor through cross-linking or some other mechanism, you might get some endocytosis that affects your results.

The mechanism (clathrin vs non) can also vary, again depending on what's being bound.

You should do some optimization - ie: do the same assay on a set of samples, with or without inhibition and see if your antibody binding is affected. But be aware that a lot of drugs for inhibiting endocytosis can do other stuff too, so watch out for artifacts. Maybe read this:

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