Last week started off with...a bank holiday! So not much science done there, and instead I spent a rainy day at Alton Towers. The rain wasn’t actually that bad, and it meant the park was pretty empty, which was a bonus – only 10 minute queues for Nemesis!
On Tuesday it was back to the lab, and I did a few bits and bobs like transforming and plating out some cells. My supervisor decided he’d like some more of a protein purifying – the HDAC binding partner we’re working on – and so we set about doing that, following much the same protocol as I talked about last week. It went pretty well, with nice thick bands of the protein being seen after gel filtration. There was one new technique we used this time though – there was a contaminating band on the protein gel, and so to further purify our protein, we used ion exchange chromatography. This separates proteins based on their different pI values (the pI is the pH at which a protein has no net charge). It uses a charged column to which the protein of interest can bind, and then buffers of increasing salt concentration are passed down the column to displace and elute proteins – different proteins are eluted at different salt concentrations and hence are separated. I took samples from the wells giving peaks on the trace and ran them on a gel, and then combined the appropriate wells and concentrated down the protein. The gel looked good, with just a single band of our desired protein.
The purification was the major work last week, and since then I’ve also started another scheme of PCR. A while ago I mentioned that I was following a PCR mutagenesis scheme to create HDAC mutants – well, we sent the final product from that off for sequencing, and when it came back unfortunately the sequence wasn’t what we wanted! So, I’m now trying that again, but this time we are going to alter the conditions slightly by using a range of annealing temperatures in some of the rounds to try and improve the output.
I’ve also been doing some catching up on my lab book as it’s the final week! I’ve taken all of the many protein gels I’ve done over the past few weeks, scanned them in to get images, and dried the actual gels down so that I can stick them in my lab book and label them. Drying involves sandwiching the gel between membranes that have been soaked in a glycerol solution and leaving for a while. It’s actually more difficult than it sounds, as you have to avoid getting any air bubbles in the sandwich, which I think I failed at with some of them as this morning I found them cracked into lots of pieces! Never mind, at least I still have nice-looking pictures of them...
So, only 3 more days to go – I’ll give you one final post soon!
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