Sunday, 25 July 2010
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.