Foreign Perspectives

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.

The morning went in preparing our samples for the PCR machine. Lots of pipetting samples in minuscule amounts, ice buckets and taking a of care to make sure our own DNA didn’t contaminate the samples. Quite a busy morning which finished with us assembling the gel for our agarose gel electrophoresis in the afternoon as it takes quite a while to set. Actually, the one consistent theme for this week seems to be that business of waiting around for something to set or be mixed and the like.

What the PCR setup sequence does is to slice out the bit of DNA that we were interested in ie CCR5-delta-32. That’s an interesting sequence as people that don’t have it are essentially immune to HIV, hence the focus on it through this summer school. Anyway, the PCR takes that bit out and produces millions of copies of it, increasing the concentration of the gene segment enough that it can be detected by the gel we used in the afternoon.

The gel process separates out the various proteins in the post-PCR mixes depending on the size of the fragments (their original shape doesn’t matter as the PCR process chops up the DNA or rather amplifies just the fragments that you want to look at). After half an hour or so it’s finished but you can’t see anything until the gel is placed under UV illumination as the dye used is UV sensitive which is a shame as you can’t see the proteins moving along the gel.

The various gaps in the day were filled in by the theory behind it all though, mainly, running behind the practical bits.

We’d our first SXR376-exclusive lecture this evening on genetic variation (the one last night on transposons was shared with SXR375) followed by the optional one on giving a research presentation.

With the change to FlyBE this year, it was well after 12 before I reached the Nottingham campus. There’s only the level 3 summer schools running this year so it was quiet the whole day.

Surprisingly the taxi was the same price as the last two years (Lenton on 0115 9 781 781 do the airport pickups for £19.50, just over half the price of the airport taxis; it’s best to arrange for them to collect you at the petrol station just outside the airport parking area as otherwise they charge for the airport parking.

Registration runs on ’til after 3pm with the introductory lecture in the biology building just after 4pm so I took the time to stock up on a few nibbles at the hospital shops (the campus shops are closed over the weekend).

It was a blue dot this year signifying that I’m in SXR376 rather than the green dot of the SXR375 people.

The introductory spiel from the admin people, learning advisor, OUSA and the course director was a joint one for both SXR375 and SXR376 kicking off just after 4pm and running on to almost 5pm. That was immediately followed by the course specific stuff in our respective laboratories. Just one lab for the course again this year as there are only about 30 of us but we’ve been split up into two groups to do the experiments over the next two days, which we’ll be doing in groups of three. For a change, we actually did some lab work this evening to get us back into the swing of pipetting.

In the room is a phone (free internal calls), wired internet connection (you need a cable for this which you can get (free) in the Cripps security office; the wifi varies from poor to non-existent in the rooms), desk, tea/coffee tray with kettle, sink, wardrobe, towels (with soap & shampoos) and a single bed. The shower-room and toilet is shared  by about half a dozen rooms. No Internet worth talking about this year as there’s some network problem that can’t be fixed until the computer people return on Monday.