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It is common practice after surface staining cells for flow cytometry analysis to wash the antibody out of solution before analyzing a sample. I have tried analysis with and without washing the antibody out and notice no significant difference by flow. So is there a reason people do this step that maybe I'm just not noticing?

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  • $\begingroup$ I'm not 100% sure but I was thinking it's to avoid getting weird background from cross-reactivity with the unbound antibodies, as in when they bind to any labelled secondary antibodies. $\endgroup$ – CKM Oct 13 '15 at 19:22
  • $\begingroup$ @Kendall So in this case there are no secondary antibodies. But if there were, I could understand, as extra primary in solution would just soak up the added secondary. $\endgroup$ – The Nightman Oct 13 '15 at 19:24
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Two reasons for antibody wash:

  1. To limit non-specific epitope binding. All ABs have specificity. Washing is usually trying to remove weaker binding to non-intended targets.
  2. For flow, excess AB that is not bound, if also pre-conjugated to a fluorophore, may allow for excitation of the fluorophore while going through flow even though the antibody isn't bound to the cell type of interest.

If you had a very specific AB, and very little excess, you may not notice a thing. I'm sure there are additional reasons; these are the two that immediately come to mind based on my own flow cytometry experiments.

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