Tuesday, 6 July 2010

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.

1 comment:

  1. why are u makinbg me read this.... its like torture for me... -___-"