13th June 2016 – Day 16

Monday:

Three extra pairs of hands from volunteers filling up the lab today and what a huge difference it made, making light work of the protocol.  If that wasn’t lucky enough, we received some new bio-bins!!! The excitement on my face when one of my colleagues told me, I laughed and asked what the occasion was, it felt like Christmas.......why she found this so funny I don’t know, all I know is that I am easily pleased and happy to have some new bins. 

In the briefing with Amy this morning we discovered that over the weekend the first protein was applied to the GF column, the fractions were collected and loaded onto a gel. Amy also purified Sufu, but she didn’t run in on a gel before applying it to the column. Both protein fractions were pooled and concentrated.

Once we had been brought up to speed on the weekends work, we set off making gels… always the first thing we do at the start of a week. 

2 setting SDS-PAGE gels

 Following that we started a pull down assay using amylose beads, purified Sufu, MBP (our control) and our 2 proteins. We are investigating whether Sufu interacts with our other proteins. We used the amylose buffers we made on Friday and collected the Flow through, the wash and the elute. This is a very similar method to purification using beads, the only difference being initially you add 2 proteins to the beads instead of one.

Each collected sample was prepared with loading dye and run on 3 gels. Once they were running, we made 4 more gels for tomorrows SDS-PAGE. With all of the additional help from the volunteers, we managed to get through everything really quickly. As a result I had some free time while the gel was running and I went down to one of the larger labs and had a chance to shadow James, a PhD student, who is working with human leukaemia cells.  It was amazing to see the culturing process which is slightly different to working with bacteria.  I was shown how things are kept sterile using ethanol, how things are disposed of and I learnt some new techniques which I hope to put to good use soon. Observing this PhD in progress using human cell lines and a new technique has cemented my career decision and passion to pursue my own PhD!! It was nice being in a different environment and being able to enjoy the nice cool air-conditioning (whilst Amy told stories of my excitement at getting new bio-bins causing much laughter).

We got the results from the gel and discovered that not much protein bound to the beads therefore next time we will prepare fresh samples of beads with the proteins and add more binding buffer as well as incubate for a longer period overnight with rotation in the fridge. 

I had such a good day today, I really enjoyed it, it was exciting to learn something new..... role on tomorrow!