Foreign Perspectives

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.

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.