We were in the cell culture room, special lab coats were needed (there were only larges left)
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.
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.
 |
| 2 setting SDS-PAGE gels |
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!
10th June 2016 – Day 15
Friday:
I
received an email from Amy saying that there isn’t much to do today and that I
could have the day off – yay for a long weekend! However Amy spent the
day continuing the experiment from yesterday, which was basically
repeating this week's protocol after the initial incubation temperature of
colonies were changed… so all in… straight forward. Amy concentrated the second
protein elute further, she applied in the GF column, same as before, 9
fractions were taken and run on a gel (so also all rather straight forward)
The
Sufu that we had harvested and frozen yesterday was re-suspended in lysis
buffer, sonicated and was purified the same as before, leaving it to be
incubated overnight at 4˚C. (Amy says she wants to come in over the weekend to
finish it off, after which she will also apply to the GF column.)
9th June 2016 – Day 14
Thursday:
I love
this picture, the bands are so clear, there is no contamination between lanes,
this is what an SDS-PAGE gel should like… the way each of the proteins have
separated by size… I am pleased with how this has come out.
Today we took
the fractions from the GF column and ran them on a gel.
Once we had decided
which fraction from the GF column contained our purified proteins, we
pooled the fractions (concentrated, quantitated, aliquoted and flash frozen).
 |
| Concentrating columns |
The elute sample of the
second protein was concentrated and stored overnight.
Once that was all
finished we prepared the two buffers which we will need for the pull-down
assays next week.
500ml of Amylose
Binding buffer
200ml of Amylose
Elution buffer
These were nice and
straight forward buffers to make, very similar to the purification buffers we
made last Friday.
It was a very
challenging day, but we got there in the end!!
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