I am having an issue with elution from neutravidin-agarose. I know my sample contains biotinylated proteins, since running an immunoblot with neutravidin-HRP shows excess signal. However, after overnight pulldown, 5 washes with 20x volume wash buffer (TBS pH7.4, 2M urea, 1mM EDTA, 1mM EGTA) and elution in an equal volume of 2% SDS sample buffer + 2-mercaptoethanol @ 95C for 15 mins I only elute two bands of protein at around 10 & 8kDa. My biggest confusion is that I do not even elute neutravidin in these harsh conditions. Looking for some advice since I can't optimize during the COVID-19 shutdown.

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