Massive Radioactive Box

Yesterday was entertaining, and I have more pictures for you to be underwhelmed by! Of course not, one of them is quite neat.

On arriving at the lab the membrane was retrieved from the cold room where it had rested in milk overnight, and we continued the washing with antibodies. As each wash took an hour I did some reading about yeast two-hybrid systems, which seem to be a pretty neat means of testing for the expression of two proteins which can interact with each other by affixing various gene activation and DNA binding domains to them. While I doubt I'll be using that in this lab it helped me understand a little more of these sheaths of papers I'm totally reading instead of going on facebook and playing endless spider solitaire.

On a related note, as well as reading up on FtsA I've been chatting to the post-grads and masters students who share working space with the Dafforn lab on occasion. As well as being really good eggs they're also working on some pretty interesting things, such as testing whether a protein is involved in oxidative stress responses by bleaching the hell out of bacteria with some peroxide. I'm pretty certain a post-grad course is for me.

Once the membrane was suitably covered in milk and protein, we turned the camera and controlling laptop on in the dark room. Here is a picture of the camera:


I've got to be honest, it does look a little bit like a coffee machine, only with more radiation hazard stickers. The lens is up at the top, and if you open the red door there's an adjustable tray on which you put your sample. An initially disconcerting number of switches on the front contain white light, UV, Power, and other such useful functions, whilst different lenses are available for different settings. We were using no lens and basic white light, which is good, as those UV-opaque faceguards looked uncomfortable.

We also made up a solution containing hydrogen peroxide which interacts with horseradish peroxidase to produce a signal detectable by the camera. After dumping this onto the membrane for 2.5 minutes Dr J transferred the membrane to another sheet of clingfilm and placed it into the camera's chamber, before taking several rather clear pictures which I shall show you later on. In the meantime, here's a picture of a tornado:


This is me making up a fantastic four litres of liquid broth, which will be used to grow bacteria in. I added 80g of the stuff and then the water, although really I should have added the flea first, as this is the bit that is responsible for the stirring. Several attacks with a long plastic rod later it was whirring around at the bottom merrily as an alarmingly deep whirlpool emerged. I had a while, so I took the above picture. All I could have added were two Galleons and the soundtrack to Pirates of the Carribean 3...

Embarassingly I mistook the foam at the tip of the funnel for some undissolved LB and Dr J had to turn the stirrer off to demonstrate this was not the case. I shall have to guard against being hypnotised by swirling liquids.

Once the broth was made I decanted it into 5 flasks and bunged them up, ready to be autoclaved. Nothing sterilises liquid broth quite like a good autoclaving, apparantly. I'd imagine a pressure cooker the size of a laundrette drum would sterilise most things.

Ultimately it was a pretty productive day; Dr J got a nice set of photographs showing his protein is being expressed nicely in his recombinant E. coli, and I got to make a storm in a 5-litre beaker. Not to mention the training for when I do all that myself.

Of flasks and horrible, horrible neurotoxins

So, what became of the bacteria I was working on half a week ago?We managed to brew up 9g of the stuff; it smelt awful. So it was placed in the fridge after being extracted via centrifuge and spatula, before cleaning several flasks. The sherberty cleaning bleach is powerful stuff but needs to be left overnight, so the next day I returned and finished the job before doing a lot of reading on FtsA. Turns out it is the CELLS that filament, not the proteins, as they replicate but don't divide properly if the divisome is incorrectly assembled. There's also lots of talk of amber mutations and two-hybrid screening. These intimidate me, so I have done a little bit of extra reading in my free time, which I shall not share with you as there is more excitement to be had.
And by excitement I mean Western Blotting.

I love jelly, so I was right at home with the SDS-PAGE (PolyAcrylamide Gel Electrophoresis). After making up the gel with such wonders as TEMED (smells like fish), APS (dangerous to touch, inhale or ingest) and water (wet, translucent) I was ready to assemble the array. After a complex procedure involving plastic locks, glass screens and electrodes, it was plugged in and Dr J and I injected samples and markers. Unlike in first year, nothing went horribly wrong and I managed to leave in the knowledge that the previous day's work had not been ruined by my previouly departed and unmourned tendency to inject into the buffer.
So once the gel was set, it was removed and sandwiched between a load of filter paper squares and sponges, inside some sort of holey thing. Refusing to be overcome by the technical nature of this task, Dr J and I ensured the nitrocellulose membrane the proteins in the gel were to transfer onto was facing the correct way, as filter paper is probably a worse adsorbant in terms of being able to wash it for several hours without it disintegrating. Then Dr J plugged in the electrodes, turned constant voltage up to 100V for an hour, and left it to run.


Now this may look mundane, but if only you knew what was going on inside that box. Sizzling hot electroblotting, baby.
There are two types of protein ladder markers, one are visible and coloured, whilst the other is detectable by antibodies and shows up with the rest of the protein you're trying to detect. The coloured markers transferred pretty much entirely, which Dr J said was a good indication of an upcoming good set of results. Although obviously there're other factors involved.

Afterwards we washed with a generic protein buffer to minimise any non-specific binding which would throw the results, a primary antibody which latched onto the tagged ZipA, and a secondary antibody which in layman's terms is shiny as hell. This secondary antibody is linked to horseradish peroxidase which interacts with hydrogen peroxide and a colourful acid to give off a signal detected by camera. But that's a story for tomorrow, as these washes took around an hour per cycle.

Meanwhile, my observation of lab hazards has been progressing. Whilst acetic acid (vingear) is to be found in various containers throughout the lab, the generic protein buffer was comprised of 1.25g of milk powder (semi skimmed) in 25g of PBS (1X). I don't like milk so I forsee no accidental consumption of lethally spiked tea. But what if you're lactose intolerant? Always wear gloves.