The third week has begun with a slow morning as Kyle came in
a little after me and Julie had to drop her husband off so she was running late
as well. But eventually we were all here
and we prepared ourselves for a busy day.
My job for the day was to prepare the cells we are going to look at
under the microscope. I had to use the
aspirator multiple times in taking the liquid out of the wells. I washed them with PBS and PBSt then blocked
and permeablized the membrane to allow the indicators to enter the cells. While that sat for 40 minutes, Julie, Kyle,
and I harvested another set of cells that were already treated. It was the same as last week where we washed
off the media and added PBS, then a protease inhibitor and a lysis buffer. Once the cells were placed in the small tubes
they were placed in the centrifuge, which only resulted in a small pellet at
the bottom. During that time as well, we
attempted to make more TBS-t, which ended up not being correct. We started to make 20x TBSt when we needed to
use the current stock solution of 20x TBSt and dilute it to the proper
concentration of 4x. Luckily that ended
up working out and we made more of the stock as well as the concentration we
needed. Once the 40 minutes were up, I
washed the cells again with PBSt and then added the two indicators to the
correct wells. We used GAP43 and PSD95 as
the indicators. Once they were incubated
for an hour I took off the antibodies and then did the same procedure again but
I put on the secondary antibody which is what will fluoresce when it is under
the microscope. While waiting during one
of the washes, one of the other people in the lab, Wade, was in today and I
learned that he is from Charleston which is where my brother is living. It was nice to talk to him about it and Julie
as well who just returned from Charleston.
Tuesday was busy as well. In the morning, I continued with the cells I
was treated yesterday. I did a couple
more washes and put on a secondary antibody again. While waiting during the longer time period,
Julie, Kyle, and I went in to talk to Kelly about what we’ve been doing. That was a cool conversation because she was
telling us where she’s hoping this project will be in the next 5-10 years. She was also discussing other projects that
have proposed other mechanisms for HIV entering the brain and the possibility
of those being accurate. One of the
long-standing controversies is if T-cells actually contain the HIV virus or if
they are just the transport into the brain.
The seemingly obvious solution would be to stop the flow of T-cells into
the brain but if we were to do that then the patient will most likely develop
PML, which is fatal. Therefore that’s a
problem in and of itself. Once the
washes were finished, the cells were ready to be mounted on the slides. At first it seemed pretty difficult since the
coverslips were in these tiny wells and so the coverslips were very small
themselves but I was able to move all of my designated ones without breaking
any. Supposedly, as Eddie said I can
probably take the title of the best newcomer to mount slides. Then we took the membrane Kyle had been
working on and we went down to the dark room to develop the film. Our first one happened to be the best so
luckily we got that. When we returned to
the lab room we ran a Fast Green analysis which is for a total protein
count. While waiting for that to dry,
Julie had begun to teach me how to analyze the western films but the Kelly came
in and told us there was another microscope demonstration that would be
starting in a few minutes. We all headed
over and that was really cool to watch all the capabilities of the microscope
especially the parts that could help me with the counting of the cells. While
we were waiting for the microscope to realign itself, the presenter asked me
how I liked working in the lab and he asked if I was on a rotation, I responded
with I really like it so far and that actually I’m in high school. He was very shocked I was so young and
working in a lab and he was saying what a great opportunity this is for me and
I couldn’t agree more.
Wednesday began a little rough as the storm from the night
before left a lot of roads closed and therefore many more people were on the
highway causing there to be a little more traffic than usual. Once I got in, Julie, Kyle, and I went into
the dark room with the microscope and were attempting to look at the cells I
mounted the day before, unfortunately the microscope needs to be serviced so we
couldn’t see much. After that we got
protein ready to be run on a gel by first making a Bradford assay to determine
how much protein was in each sample so we could make sure that each had the
same amount of protein once we added the water and loading dye. We used a spectrophotometer which I have used
at school but this was more intricate than ours since there was a special
program to compare it against the standard curve and then it’s called a nano
drop because you can put a small amount of DNA or RNA on a small plate and it
will tell you how much is in that sample.
After we got the solution prepared to be loaded onto the gel, Julie
allowed me to set up the gel electrophoresis and while that ran the three of us
went down to the dark room to develop the membrane Kyle has been working
on. That ended up being really
overexposed so we had to do a couple more washes. By then the gel was almost done running so
Julie and I got the membrane and all the things needed for the transfer
ready. Then I got to take out the gel
and make the “sandwich” to transfer the protein from the gel onto the membrane. While the membrane was washing Julie took
Kyle and I out for gelato since it was Kyle’s last day and Eddie tagged
along. It was AMAZING. Supposedly it’s the best in the world or the
best in the country, I don’t remember which one, but I got dark chocolate and
hazelnut which was one of the hardest decisions to make. We sat outside and talked for a while, which
was very nice and relaxing. Afterwards, Kyle went back to Julie’s apartment
since he was staying with her and her husband and that was the last I’ll see
him. He is going to Vietnam this coming Friday,
the 26 to teach English which is incredible.
Then we walked back to the lab and took out the membrane that was in the
transfer and got that in the BSA to wash and the other membrane sat in
TBSt. During this entire time, Eddie has
been planning parts of his wedding so that has been entertaining for everyone
else in the lab, so we had all been helping him plan his rehearsal dinner. Once the washes were finished we tried one
more time to see if there was protein on the older of the membranes but the
film didn’t show anything, we used Fast Green and even then there wasn’t much
total protein on the membrane. We put
the other one in the primary antibody and let that sit in the cold room
overnight.
Thursday was a good day.
I got in at 9 and Julie and I started with the membrane. We began the washes and while that was going,
the proteins for another gel were thawing.
I prepared the Bradford assay to determine the protein concentration in
the samples, same as yesterday. After we
used the spectrophotometer and determined the proper amounts for the components
to reach the same protein concentration, I mixed everything together. At first I had read the pipette wrong and
instead of putting 23ul and 28ul, I put 2.3ul and 2.8ul. Luckily that was an easy fix and I just had
to add more but it’s a good thing I caught my mistake before we added the
protein. The secondary antibody was
added to the membrane that was washing and the protein was heating up. While we were waiting for that to finish,
back in the “office” Eddie, Anna the newest addition to the lab, Julie and I
were talking about lab efficiency and got on the topic of diapers in lab. That was very funny and then the case of the
woman who drove across the country in a diaper to take revenge on a woman came
up and we were cracking up about that.
Being able to openly talk about weird stuff like that is so much fun. Then Julie got all the stuff out I would need
for the afternoon because she had an interview she had to go to. I’ve been doing this on my own which is
pretty exciting, I feel very independent but I also have many others in the lab
like Eddie and Cagla if I need to ask a question. I finished the washes and put the substrate
on the membrane then got everything ready and Eddie and I went down to the dark
room. I developed a couple different
films in hoping that the background would fade and the contrast would be
greater but I needed to do a couple more washes. Unfortunately, when I went back down, nothing
really showed up so I went back up and put the next primary antibody on and set
it in the cold room. There was a lab
wise meeting at 3 but I didn’t need to go to that and luckily my mom had to
pick me up then anyway so everything worked out perfectly.
Friday began well.
The drive in was so easy, no traffic whatsoever. When I got to the lab, Julie had already
started the washes on the western and running the gel. I put the secondary antibody on the western
and let that sit for an hour. I cut the
paper and the membrane for the gel, afterwards.
Once the gel was finished running, we made the “sandwich” with the
sponges, papers, gel, and membrane and set that to run for an hour. We began the second set of washes for the
western and while we were waiting for all of that to finish, Julie and I were
talking about the group of students that will be attending our lunch next week
and began talking about Peddie in general and all of the international
students. Then college came up and
boarding life which was interesting to hear her opinion. The membrane was finished transferring so I
took apart the setup and extricated the membrane then put it in BSA to block
it. Blocking is when you cover the
membrane so that only the proteins show up when developed, otherwise the
antibodies would stick to anywhere on the membrane because it’s sticky. That sat for an hour so we ate lunch. That was fun, there was a group of us talking
about vets and dentists. Once that was
done, we set the first membrane to go through the next set of washes before
developing it and the most recent one was placed in the fridge in TBSt. Julie had to leave to go to a wedding so I
was on my own for the last two hours of the day, but it was something that I’d
already done numerous times before and had no problem by myself. The only problem I ran into was that when I
developed the film, it was either extremely dark where you couldn’t
differentiate between anything or extremely light where it just didn’t show
up. This was quite frustrating since it
occurred no matter what time I laid the film on for or if I had a filter. I went back upstairs and Julie said to just
put it in the fridge with TBSt and she’ll add the primary antibody on
Sunday. For the last couple minutes of
the day I just relaxed and read some more of the grant proposal Kelly had given
me. Finished with my third week already,
half way done and it flew by.
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