Rain, ITC, Rain, Swing Dance and Rain

So, it's been, uh, raining, in Newcastle.  Not that I'm surprised by this fact, though I was hoping for slightly less wet weather in July.  Sorry for the lack of blogging.  It's not for lack of results I assure you, but more from the catching up needed when moving house as I have done.  Not quite managed to fully settle in, but that's another story.

So, what I've been doing for the past two weeks, is testing the interaction of my protein with a bit of E. Coli.  We figured out that my assays (where you grow up the cells and add the protein and end up with a pretty blue gel) might be binding to a different part of E. Coli (OmpF) to the bit I want to look at.   Because of this, I did my experiment again, with the different cell mutants, and then added a mutated version of my protein that doesn't bind OmpF to see what happened.  What happened was, nothing at all.  I don't know why, but either that protein isn't concentrated enough, or something else happens, because it doesn't show up on my gel at all.
I may try remaking the gels with a different stacking buffer, as the new stacking buffer I made I tried to pH with diluted acid.  Having forgotten one of the fundamentals of A level chemistry, my buffer, well, buffered the acid.  I eventually found the glacial HCl under the fume cupboard, but that was about 70ml too late, and in a 300ml solution, I think that really didn't help.  Anyway, I tried the whole thing twice and it didn't work, so if I get a chance to make up some new gels, I have my samples in the freezer, so that should be ok.

The main thing I've been doing is ITC.  This stands for Isothermal Titration Calorimetry.  Basically, you put the two things you want to test the interactions of in the same buffer.  You do this by dialysing one protein, then making up the other sample in the solution that you dialysed the protein in.  You then put one thing in the syringe of the machine and the other in the cell.  The machine then injects the contents of the syringe into the cell a bit at a time, and measures the heat exchange, and you end up with a nice peak on a graph.  The heat exchanges are supposed to start big and get smaller as the number of possible interaction sites gets smaller.  The heat exchanges can either go up (exothermic) or down (endothermic).  Mine appear to be going up, which apparently means that my interaction A. takes place (YAY!) and B. that it through hydrophobic binding, which is also good.

I tested my full length protein then different combinations of domains.  There are three domains.  Between two sets of results I found out that one part of my protein binds what I'm looking at.

The first few rounds I did this,  I only got some very little peaks on my graph, so it was suggested that I try the interactions at different temperatures.  I got some very good heat exchanges at 38 degrees (C), but my protein is unstable at that temperature and seems to precipitate out half way through the cycle, and give slightly strange results at the end, so that was out.  25 degrees, which is what I started with gave better results the second time round, and my protein is stable at that temperature. I think I might have made one of the solutions up more concentrated, which will have helped my results.  At 12 degrees, I got some good results, small, but not insignificant peaks on my graph.  However, when putting protein into buffer, as a control, my peaks started off small and got bigger.  I have a feeling that my protein interacts with itself at 12 degrees.  The graph looks mightily strange compared to the others!

I'm now testing different lengths of what I'm adding my protein to to see what happens there.  The results are looking nice, though I panicked this morning when one of my graphs decided to have no heat exchanges at all.  Either there was something wrong with the experiment, or I accidentally put the wrong thing in the wrong bit of the machine.  Likelihood is the latter, but the other two runs turned out the same, so I will put that result down as an anomaly.

I've been surprised how excited I've managed to get over what are essentially graphs of two solutions getting minutely hotter or colder, but when you start getting results that you hypothesised were going to happen (seem to be reliving A Level statistics today), it's hard not to get excited.  Trying to resist the urge to tell everyone and distract them from their research is quite hard, but there are a few people who are doing research directly related to what I'm doing, so they have a vested interest in what I'm doing.  The lab is quite friendly though, so people will usually chip in on conversations you're having and make useful suggestions or want to know what you're doing.  I think I've been blessed with being in a particularly friendly lab, and I'm very glad about it!

Between all of this, I've been enjoying the sites of Newcastle, whilst listening to swing music, and managing the odd spot of social swing dancing, which has been lovely.  The quayside in Newcastle, looking over to Gateshead is utterly idyllic.  The combination of the regeneration, which is very smart and modern, with the old bridges and pubs and art studios in old buildings gives an amazing atmosphere, and despite the rain, there have been a few beautiful evenings perfect for the odd dance down by the river.

One last thing.  In the lab.  Never bother to wear anything less comfortable on your feet than your MOST comfortable pair of shoes.  The pain in your feet from standing up all day genuinely is not worth it.  Lab coats and trainers all the way.  :D