Crashy crashy spin spin bang bang die

Today I tried my hand at ultracentrifugation. ANALYTICAL ultracentrifugation.
The difference between the two being that a normal ultracentrifuge spins samples at several hundred thousands of g's, whilst an analytical centrifuge does so whilst lasering the hell out of the samples. Check this out:
I'm sure many of you are mistakenly wondering why I've photographed a tumble drier. This thing costs about £250,000 and as such I wasn't allowed to operate it due to my lack of training. It costs labs £100-150 to use it and typically runs for over 12 hours straight, but the last use clocked in at 21 hours 24 minutes. Analytical ultracentrifuges can tell you all sorts of neat things (via lots of complicated maths) such as dimeric/trimeric etc. states, sedimentation coefficients, and whether your sample is in fact protein or, somehow, lard. I enjoyed a 2 hour demonstration by Rosemary, the lovable yet endearingly furious lab technician who was very enthusiastic about the whole peice of kit. Then I got to assemble the cell:

The sample goes into one of those holes, with some buffer as a reference. The transparent discs are made of sapphire, but the centrepeice is the expensive bit. The whole thing is worth three figures. I tried very hard not to drop it.

Sadly I misunderstood the necessary ingredients I would have to supply. "400 microlitres of sample and 500 of buffer" caused me to dilute my proteins almost out of existance and the signal was so weak a reprisal is necessary. Damn.

In other news, I added a mystery reagent to the mixture of membrane proteins we were binding to the resin overnight for the column elutions. Once bound to the column I pour the elution buffers through to get most of the useless proteins out before my protein washes out. However, this mystery reagent royally buggered up the binding mixture and I had to dilute it out by a factor of 1 in 4. So with two 60ml samples and a lot of precipitated gunk blocking the column, there was only one way I was going to finish before 6pm.


CAN YOU EVEN BEGIN TO UNDERSTAND WHAT YOU SEE HERE? Richard, in the other lab behind two sets of fire doors, had to sit down due to all the excitement in the department. Also he was doing lots of pipetting and his back would hurt otherwise. But seriously, despite the flow rate of one drop about every 10 seconds, it was unspeakably hectic.

I left the lab at about 6:20. Tomorrow I shall be doing Circular Dichroism with Dr Dafforn (hopefully), and shall be sure to attempt to avoid bursting the 5ft tank of liquid nitrogen in the process.

Two at a time

Just thought I'd show you this: it's a heat block! I have to break apart liquid cultures so their contents will leak out and be detected in the SDS-PAGEs I do. So I usually set it to 98 degrees and cook them for 10 minutes. I don't see the point in the warning on the top though..."Warning - the block may be hot!"

It's a heating block. That is the absolute MINIMUM I'd expect.
So last week!
I performed a western blot on all the cultures we'd grown in order to make some FtsA recently. The small-scale cultures were in a nice little lunchbox-style array with several wells and a squidgy silicone lid. However, there were 16 wells with bacteria living in them, and one SDS-PAGE can only hold 8 lanes, as two have to be reserved for protein markers, which we can use to compare the bands we get to bands of defined mass!
So I had to do two at the same time. It was intense.




You can't tell very easily because the bubbles produced when the power's turned on are obscuring it, but THERE ARE TWO GELS IN THERE. It's also nice to check back occasionally on the apparatus to make sure I've connected the electrodes the right way round, and that the band isn't currently travelling upwards, which would be bad.


So after that had run, I took out the gels, sidled them with a nitrocellulose membrane, wrapped them between sponges and filter paper, and stuck them into the buffer. But unfortunately whilst I was otherwise engaged I forgot that I'd run two at once. So when I opened the lid after an hour I saw this:


Me: "OH IN THE NAME OF EVERYTHING THAT IS HOLY, THERE'RE TWO OF THEM!"

I almost soiled myself. However, composure and the ability to cope with previously unencountered phenomena can make or break a scientist, so I calmed down and adapted, excising the membranes and placing them in blocking buffer, antibody, SDS-Tween and then another set of buffer and antibody:



THERE ARE TWO GOING AT ONCE! CAN YOU EVEN BELIEVE?

Ultimately it was a challenge in terms of organisation and dexterity, but it worked well ish. The antibody we used was new and may have had some teething problems. However, despite this there was a signal present, albeit a rather weird one. So I'm running a comassie stain which will hopefully shed some more light on this quandary.

Oh and also I got to mess around with some liquid nitrogen, but that's really boring, so I didn't take any pictures.