Anyone have ideas on what this blue band could be on this Q sepharose column from C. difficile R20291? Soup was ran through thyroglobulin column to pull out TcdA and then through the Q sepharose with 20-80% NaCl gradient for 2hr 🦠 #microsky - ThreadSky

anniedoyle.bsky.social • 33 days ago

Anyone have ideas on what this blue band could be on this Q sepharose column from C. difficile R20291? Soup was ran through thyroglobulin column to pull out TcdA and then through the Q sepharose with 20-80% NaCl gradient for 2hr 🦠 #microsky

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dkountz.bsky.social•33 days ago

Did you grow with resazurin in the media?

anniedoyle.bsky.social•33 days ago

Nope! I feel like maybe this could be a copper containing protein maybe? I’m keeping the column in the cold room for now because I want to go back and try to figure this out.

dkountz.bsky.social•33 days ago

Fwiw I’ve seen multiple anaerobes that produce blueish (presumptive) proteins that elute very late on q seph columns. Got to be a copper protein. Maybe cu/zn superoxide reductase

anniedoyle.bsky.social•33 days ago

I have a strong feeling that’s exactly what that is. For growing native toxin we actually don’t grow it in the chamber but instead boil, purge, and stopper 4L flask then inject culture into large amounts of hanging dialysis tubing and harvest after a few days

anniedoyle.bsky.social•33 days ago

But we don’t gas exchange with a manifold like I have with acetogens, so there must be a microaerophilic environment at the very top of the dialysis tubing, most likely stimulating the superoxide dismutase

anniedoyle.bsky.social•33 days ago

To be 100% honest, I really don’t consider C. diff to be a true anaerobe but rather an aerotolerant bacteria since it doesn’t immediately die when oxygen is a mile away like most strict anaerobes I’ve worked with… maybe it’s just extra good at making superoxide dismutase!

myonlyneuron.bsky.social•33 days ago

Annie, Not to be a smartass, but that blue line is an indication that you need to focus on REAL science that requires microscopes.🙂

dkountz.bsky.social•33 days ago

Tbh, I’ve noticed that most “obligate anaerobes” that grow relatively well just don’t want a full oxic atmosphere. Even then, they’ll put up with it for a long time and just stop growing for a while

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