Tuesday so far has not gone as planned. I was expecting to be able to start by
putting the primary on my one blot but the problem I faced was that the
antibody had not come in yet. Julie
ordered it before she left so it should have arrived but no one received any
antibody packages and no one can find it. I’m hoping that the company is
running behind or hasn’t shipped it yet instead of us receiving it and getting
lost in the lab. Anyway, Julie told me
to skip it and go to the next primary. So
I put on the PSD95 and hopefully the Axin1 antibody will show up. With the other blot I did a FastGreen to see
the total protein on the blot. I had to
make more of the destain so I got to use the methanol and acetic acid so I was
happy to do that. Then while the primary
was incubating, I worked on my blog post and my poster. Julie had said that my intro looked very good
and made sense and was accurate so now all I really have to work on is the
method because we ended up doing something different than I was told and the
actual results. I went out to lunch with
the now Dr. Venanzi and Ms. Cozine along with Matt, Emma, and Julius. That was a lot of fun, we went to Wahoo’s
which I had never heard of before. Then
afterwards, there were cornhole boards outside so we started tossing some bean
bags while Dr. Venanzi and Ms. Cozine were taking pictures. The trek back was long and hot but Matt and I
did it safely. Tomorrow, Dr. Venanzi and
Ms. Cozine are coming in again and we are going to a place called the White
Dog, a couple of the others were saying it’s really good so I’m looking forward
to that. I finally got back to the lab
and the paper for the Axin1 delivery was on my bench! That was very exciting because now I don’t
have to worry about not having it and what will happen once I leave. So I got back and then began the washes on
the blot I had put the primary on earlier.
It was later than I thought and I was thinking about what I should do so
that I don’t keep my mom waiting for too long outside. I finished the washes and went down to the
dark room to develop my film. Luckily
the signal was very strong and I only had to lay the film on for a couple of
seconds and the bands showed up well. I
got back upstairs, put the Axin1 antibody on and set it in the cold room
overnight.
Wednesday started with a nice ride in with Dr. Venanzi and
Ms. Cozine. We were talking about a lot
of different and interesting things and laughed a lot. Once I got into the lab I started to run a
gel. I had a couple questions to ask
Eddie and Anna but it ended up working out well. I loaded the gel and let that run while I
prepared for the transfer. I set the
transfer up after the gel ran and let that run while I went out to lunch with
all the other EXPers. The lunch was a
lot of fun because I got to talk to everyone and the food was really good as
well. We all walked back to our
respective labs and once I got back the transfer was still running but I had
set it to run longer than I needed to make sure that it was ok when I came
back. I took that out and blocked it
with BSA. I started the washes on the
other gel I had set in the cold room the night before. When I was done with the first set of washes
and had to put the secondary on, I didn’t realize I didn’t have enough BSA to
both block and make the secondary.
Luckily the blocking was finished at just about the same time as the
other blot so I could reuse the BSA that was used for blocking and then make
the secondary. While that incubated, I
scanned in the films I had done the previous few times. I hadn’t had time to scan them in when I
completed them so luckily I had time today to get them into the system and
quantify them. When I was done with the
second set of washes, I went down to the dark room and developed the
films. Once I was finished I came back
up and put Axin2 on because the last time we tried it, it ended up being dark
and difficult to quantify.
Unfortunately, the same band has been showing up on the previous three
blots so that one, synaptophysin, must have a really strong signal. I put the Axin1 back on the blot and
hopefully if I expose it for longer the other bands will appear.
The ride in on Thursday didn’t go quite as planned. My mom’s car started to give her trouble when
she ran out to get something and so we had to go get my grandmother’s car which
was north. Then we got on the turnpike
but she started to head north instead of south.
Luckily, we were able to turn around at the next exit and go south. Once I got in, I started on the washes for
both blots. I asked Anna and Eddie a
couple questions about my project but the main problem is that they are not
familiar with this project and therefore can’t accurately answer my question
unless they have more information, information I don’t know. Hopefully, I’ll be able to figure it out and
Julie can help me but I’ll do my best. I
put the secondary on both blots and I had to be careful that I put the correct
one on the correct blot. I did that and
did it successfully. I finished up with
the washes and then went down to the dark room.
The blot that has been giving me trouble still gave me trouble, the
protein I wanted, Axin1, didn’t show up only the synaptophsyin did. But the other blot, that I ran the gel
yesterday, worked really well and the PSD95 showed up nicely. I was very happy with that and it made me
feel better that one actually worked. Then
there was a meeting that I didn’t have to go to so I was able to leave
early.
Friday. My last day
as of the moment. I brought in donuts
today because I am planning on today being my last day. I will be done with everything Julie gave me
to do after she left so unless Kelly needs me to do something else, I’m
finished. I came in and started to work
immediately. I grabbed my blot from the
cold room that was incubating in primary and I started the washes. For the other blot, I decided that no matter
what I’m doing the same protein is showing up so I ran a FastGreen to determine
what the total protein is and make sure I’m not going crazy. I put the secondary on while the destain was
on the other blot. I’m hoping the
FastGreen worked decently well or that it shows the only protein that’s
prominently on there is the synaptophysin that continues to show up. I then started to look for the file that has
all the pictures I took a while ago and it wasn’t on the computer I needed it
to be but it was still on the computer in the microscope room. I asked Anna if she could help me and she
asked Cagla if we could use her flash drive which she let me use it. So I’ll have all those images on the proper
computer so I can then analyze the neurites.
I did the last three washes on the remaining blot and then went
downstairs to the dark room. Luckily,
the antibody worked well and the bands showed up nicely. When I got back upstairs, I didn’t have any
more proteins to probe for on this blot so I decided to run a FastGreen to
determine the total protein concentration.
While I was waiting I got a response from Kelly and we will meet next Tuesday
or Thursday to discuss my results and what I’ve learned and what my career
plans are. While the blot I FastGreened
dried, I counted neurites on the neurons I had taken images of a little while
ago. Seems like an appropriate way to
end my time in the lab, I started with counting neurites and I ended with
counting neurites. Things really come
full circle.
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