A bad day in the lab – science never goes as expected!

I promised to post again today with an update on in-house screening of my hit for fragment screening, but sadly, there’s nothing to update, as yesterday turned into a litany of disappointment (not to be too much of a drama queen!). First, I started a fresh purification of ACVR1 to prepare for setting up new plates for XChem. So far, so standard. However, the centrifuge tube decided to split and spew several mls of my lysate into the rotor during centrifugation. I salvaged some of it, as I need every single microgram of protein possible, but that was a bit of a mood-changer on a beautiful sunny day in Oxford.

While the supernatant was incubating on the nickel resin, I went downstairs to mount my beautiful new LDN crystals for in-house screening, just to see if I could get reasonable diffraction and potentially index the spots to get the space group, as we’ve had a variable space group problem with the other condition I’ve been using – not very good for the bulk processing programme, PANDDA. I opened the well, pulled back the skin on the drop, got the cryo-protectant ready, went to scoop a crystal – and they all, all 7 of them, disappeared before my eyes! Dissolved! Tiny bits! Disaster! I managed to mount a fragment, and Beth, our lovely crystallography engineer, put it on the Bruker, to no avail. No diffraction. Sigh.

So I went back up to complete my purification, ran the gel of the nickel fractions, and there was absolutely no protein. Clearly something went wrong with the expression.

At this point, it seemed a good time to pack up and go home, worrying all evening about how I was going to get protein to make crystals in time for the fragment screening trip, since the biotech insect cell pipeline can take a couple of weeks or more depending on how many requests they have. However, good news! I spoke to Shubhash, our biotech pro, this morning, and he is putting in an urgent request for cells for me (he said he had a couple of litres spare), and I can harvest on Monday. He has checked the latest test expression, and it was good, so it looks like all might be well, if I get a move on and purify straight after harvest on Monday. Shubash, my hero, saves the day!

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