Foreign Perspectives

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.