Foreign Perspectives

With several groups working well into the evening yesterday, the conclusions team was probably not the best team to be on.

Some last minute discussions on our immunocytochemistry presentation resulted in a few updates to what we said but, for a change, no last minute changes to the text of the presentation itself. Unlike last year, we all had at least some knowledge of the various topics being presented which made for a more interesting feel to the morning I think. Once they were completed, we even had some feedback on how well we’d done or rather on some improvements that we might make for the next time.

And then it was time to go our separate ways.

It took a little longer than expected to reach the train station but I’d still got loads of time to wait around. The first class ticket was definitely worthwhile and the meal broke up the journey quite nicely. As it turned out, it was just as well that I took it as there was nothing open on campus by the time I arrived.

Although, at first glance there seem to be cafés and restaurants everywhere on the York campus, outside term-time the opening hours are rather limited. On Friday night there’s only one open and it closes at 7pm. The supermarket in the market square is open Monday to Friday 9am to 5.30pm with the little shop beside it only open from 9am to 2pm Saturday and 9am to 1pm Sunday.

In principle, the remaining lab work looked like it would only take an hour or so but in practice the final washes and particularly getting the slides mounted took ages and we didn’t complete our photography of them until lunchtime.

That in turn meant that we were running rather later than expected in starting the preparation of our presentation for tomorrow. So late that we have one final image to be inserted and we’ve not yet had a full run-through. Mind you, we are the penultimate presentation so we may have time to polish it up a little more before we’re on (although probably not if last year is any guide).

Anyway, that’s the lab work done for this course and we’ve only to do our 10 minute presentation tomorrow before we all go our separate ways. Well, not quite all as a fairly large number of people seem to be doing two residentials this year.

It’s off to see about reading up on my section of the presentation now (short, but with lots of potential for questions).

The final thing we’d done yesterday was to pick the “further experiment” that we’d be doing today and tomorrow. There wasn’t nearly as much chaos in that as expected and almost everyone got their first choice of experiment.

Work on our immunocytochemistry project began quite slowly as such things tend to do but before long we were into the swing of preparing quite a number of different reagents over the course of the day and applying the various blocking solutions, primary antibodies and secondary antibodies. Much the same as we’d done earlier but with different antibodies and blocking solutions as we were aiming to find out if CCR5 is expressed on the surface and not inside the cells.

As with yesterdays washing exercises, nothing was to be visible until the very end of the day when the colouring enzyme was applied. It turns out that this must be prepared freshly (even 10 minutes is too long for it to sit) so one group had a minor panic when nothing happened. We thought that nothing had happened either but hadn’t thought that since we’ll be looking for the effects in a microscope, that the cells were pretty small and in fact looked like there was some dust at the bottom of the well.

There was a fair bit of reshuffling of groups this evening as we’ve eight topics to present on Friday morning, 32 people to present them and we were all in groups of 2 or 3. It all worked out quite quickly though and I’ll be part of a slightly larger group presenting the cytochemistry project which is the penultimate one (not a great position).

The final tutorial was one aimed at both SXR375 and SXR376 though all but a couple of those attending were from SXR376. It was on the end of course assessment and seemed rather more general than the, supposedly identical, tutorial on the topic that I’d went to for SXR375 so I’ll have to hunt out my notes from that one when I get home.

Today picked up where we left off yesterday.

The deep blue coomassie stain that the gel was floating in needed to be washed off so that we could see the bands made by the various proteins. Although this is, in principle, fairly easy to do it takes quite a number of washes before the bands appear clearly. What this does is to allow you to estimate the total amount of protein captured in the gel so you can in turn estimate the amount that’s actually due to CD4 and CCR5 from the nitrocellulose membrane.

Whilst that washing was going on, the nitrocellulose membrane was being passed through a separate series of washes starting with a primary antiblocking solution before treating the CD4 and CCR5 in anti-CD4 and anti-CCR5 antibodies to bring out the CD4 and CCR5 bands for identification.

Both these took ages to do with a number of changes of reagent followed by mixing stages that ran up to two hours. Then there was even more of the same but with a secondary antibody before we finally added the HRP to colour the bands. The big worry about this type of experiment is that you can’t really see anything happening until the end: a bit of a problem if you’ve done something wrong to say the least.

Once the bands are coloured, you can work out the relative molecular masses and thereby identify the proteins.

Amazingly, everything worked really well for us and we even came up with the right results and a fair bit ahead of time.

The evening “tutorial” was pretty much our own production. When we arrived our groups were shuffled a little before we were given 30 minutes to prepare a talk on a series of topics covering all the work we’d done in the previous couple of days. It turned out to be a fantastic way to revise that work – certainly a whole lot better than have the tutors do a recap of the work as usually happens in these things.

On to SDS-PAGE today. That’s another gel but a polyacrimide one this time which means a bit of fume cupboard work. It’s more complicated to assemble this gel as it’s run vertically so there’s a bit of assembly work to be done then you’ve to use two gels, one to actually run the analysis and the other to make sure that all the samples start off at the same time.

Things went rather well and we’d a relatively early finish to the lab work this afternoon. The downside of today is that we finished with nothing actually visible.

The gel was processed in a sandwich to move some of the proteins onto a nitrocellulose membrane with the encouragement of a little electricity yet again. With that done, processing of the gel and the membrane separates with the gel going into a coomassie blue bath to visualise the proteins whilst the membrane is cut in two so that the CD4 and CCR5 sections can be processed separately tomorrow. For today, the gel is washed in the coomassie whilst the membrane is washed in western blot blocking buffer.

The evening lecture on the biology of HIV was fascinating though it would have been better to have it earlier in the day as there was an awful lot to take in at that time of night. The optional lecture on how to write the research plan was useful and fairly short too. Those that didn’t go to it will regret that when the marks come out.