Wednesday:
Already half way through
the week and it still only feels like its Tuesday. I arrived at the lab this
morning only to hear that the blots hadn’t transferred, this is so frustrating
but I guess it is the nature of the game. After looking at them we decided that
the SDS-PAGE gel might have been left in the transfer buffer too long, the
protein appeared to have diffused out of the gel and therefore we chose to
abandoned these blots and not take them any further.
Luckily we don't throw
away our samples until we are one hundred percent sure we no longer need them
and funny enough we forgot to put them into the fridge so they were still
floating about… literally floating on what used to be ice. I fished out all of
the tubes and re-did the two gels from yesterday, without the beads this time.
(The one for staining and the other for the western blot).
I cracked on with this
while the other three volunteers carried on with pull downs assays using nickel
beads and three different protein samples, from samples that were previously
harvested.
It was a slow paced day
but between us, we achieved a lot. The other three finished the pull downs and
got them ready to transfer to a gel, which will be done tomorrow.
Everyone else left but I
stayed on, I wanted to carry on with other things during the hour long
incubation steps. I made all of the buffers that I would need. I do enjoy
making blocking buffer (who would have ever thought about using milk powder in
a western blot, genius!). I made some more de-stain for the gels, I got so
excited when I was told I could do this, more clean fresh de-stain. It’s so
transparent, no dye in it yet...... I suppose it’s like the bio-bins all over
again..... can't help myself.
I started on with the
western blot on my own and finished at 6 pm as anticipated, and
really proud of myself. I carried out a western blot from start to finish all
on my own, no mistakes, and finished when we had planned. So a good day all in
all…. Well except for when I dropped the transfer buffer upside down into the
tub upon where it all emptied out and almost went everywhere ...... but
excluding that then.
During incubation time
of the antibodies on the membrane, I had a chance to take some photographs
of all of the other gels, of which there are many. It is easy to get them
confused, but I was careful to ensure to label the boxes as we go.
Once the western blot
was completed, we took a photograph of it, sadly it didn’t show much in the
elute fraction with the amylose beads. It was very disappointing to see, well
technically not to see, but we definitely know it worked because the histag
antibody attached to the marker which has showed nice and clearly.
I’m really pleased to be
home filling in my practical notes and just preparing mentally for tomorrow, I
will be doing another western blot…cross fingers this one actually shows us
something of use.
During the morning catch
up with Amy while we were discussing what we were going to be doing for the
day, I saw out of the corner of my eye, someone sneak into the lab and nick one
of our new bio-bins, I realise now that perhaps we shouldn't have flaunted our
wealth quite so much. Haha but at least we know where the new stash is being
kept.
