el blog de Brian Gilmore

Week 5

2005-07-15T06:42:00.000-05:00

Well, we're coming down to the wire here in terms of finishing up my experiments for my presentation at the Symposium as this, the penultimate week, wraps up.

The first few days, all I did was prep and run an experiment. Simple enough, as I've done it countless times before. I (in some cases we) split cells, treated cells, collected lysates, poured gels, loaded gels, ran gels, performed a western blot, treated the membrane, so on and so forth. This experiment finally left me with some usable data in terms of what I can put in my presentation.

Thursday, things got hectic. The data which was collected Wednesday needed to be followed up on by another set of experiments. This, along with the stages of two experiments that will be run in the next few days. This had several experiments occuring all on one day. I was on my feet almost constanly, finishing collecting lysates here, exposing film there, taking pictures of cells there... Certainly an eventful day.

Even though I'm staying late today, and possibly Monday and Tuesday, it's really going to be a rush for me to finish my presentation and experiments by Wednesday morning, but with luck (and Dr. Kridel's help) I should be able to manage it.

Week 4

2005-07-08T06:54:00.000-05:00

The 4th week was a short one, only 3 days in-lab due to the 4th of July. Though I enjoyed the day off, this did leave me with 4 days of work to do in an actual timespan of 3 days.

Tuesday was incredibly busy. I did three Protein Assays, loaded and ran two gels, performed two western blots and collected a set of Lysates. That means I was running two experiments simultaneously. This was the only day I had to do this, because the results of my third Protein Assay showed that there were problems somewhere in the experiment (possibly in the preliminary stuff that I had no part in) that spoiled both our controls, and rendered the experiment unusable.

Wednesday and Thursday were uneventful. I treated the membranes from Tuesday once each day (peIF2-alpha on wednesday, and total eIF2-alpha on thursday), but this left me about two hours of work to do, and the workday is much longer. So during the down-time I helped our grad-student, Joy, with several of her projects. This involved collecting Lysates (a set of 8 and a set of 12) and doing some tissue-culture. This also got me a crash course in ER (Endoplasmic Reticulum) Stress and how it applies to cancer.

So the fourth week was short and uneventful, but well spent.

Week 3

2005-07-01T07:25:00.000-05:00

Week 3... Halfway done...

More of the same this week, with the exception that my work actually related to the presentationI'll be giving at the Symposium. The big thing this week was doing a pair of experiments from front to back (meaning all the way from Tissue Culture to Membrane Treatment) at the same time. Though it actually ended up being more like 1 1/2 experiments.

The first experiment went well, and the data was what we were expecting (maybe "hoping" is a better word...) and our second treatment designed to check for accuracy of the other treatment came back with excellent results. We even got to messing with the standard procedures a bit due to how well it was going. The second one, however, taught me the pains of research... the hard way.

The Tissue Culture, in which we treated several plates of LNCaP (Prostate Cancer) cells went well, and we let them incubate for several days. The lysing and collecting the lysates went well. The gel setting, loading and electrophoresis went by without incident. Then came the western blot. I loaded the clamp backwards, meaning that I put the gel on the wrong side of the membrane. Such a simple, stupid mistake messed up and entire day's work.

And then, it gets even better: I tried the experiment again the next day, and got through it without incident, except for one little problem. We didn't have enough sample to actually do the entire experiment. So our results were muddy, unclear and didn't help at all. Well, at least I won't be making that mistake again, and as Dr. Kridel says, "worse things have happened in the world." I just have to do the experiment again next week.

All in all a pretty good week, despite that small problem at the end.

Week 2

2005-06-24T08:24:00.000-05:00

One third of the way through the program already...

The second week has been a lot like the first. I've done primarily Western Blots (4 this week), a pair of Protein Assays, and an ungodly amount of membrane work and film exposure. My work is now usually fairly independent, but I've always got help at hand if and when I need it. My pipetting has improved immensly, both in speed and accuracy (best correlation coefficient this week was a 0.996, when last week it was 0.973), and I'm finally starting to get the hang of being and working in a darkroom.

One new thing that I got to do was some Tissue Culture. TC involves any number of things done with some of the stock of tumor cells we keep incubating in the lab. This week, I split some cells in the biological cabinet, meaning that I took some cells from one plate, and put them onto several (4 each from 2 of them, in this case) others. Then, to test a hypothesis derived from one of my film exposures, I added varying concentrations of Apigenin to 4 of the plates (keeping one as a control), and waited to see the effect. It seemed to me that Apigenin was effective in killing off the tumor cells, though this may not mean that it is the way to cure cancer, for a variety of reasons.

The last thing I did was to check the [lysate] of the apigenin-treated cells (and control) by aspirating the media, adding PBS, centrifuging them, aspirating the PBS, lysing them, letting them centrifuge in the cold room, then removing and saving the supernatant while discarding the cells themselves.

Another fun week draws to a close. Next week, real work towards my hypothesis and presentation should begin.

My first week

2005-06-17T08:07:00.000-05:00

Well, the first week of the CERTL Program is drawing to a close. Given that it was the first week, a lot of my time was spent going over the basics under close supervision (not that I'm complaining, I don't really trust myself with most of this equipment)

But, that's not to say that I didn't accomplish anything. Dr. Kridel has spent lots of time right there with me, making sure that I can handle anything, and making sure that I understand what's going on, not just how to do it. Joy (who was gone until thursday) and Frances are both great at explaining things when I'm lost or confused, which happens to be a lot of the time.

With their help, I've learned how to do Protein Assays, in which we serially dilute proteins, then essentially dye them, and measure the color with a computer program and machine to determine their concentration. I've done a pair of Western Blots, which involve mixing and puring gels, adding protein samples, and then seperating the proteins by molecular weight inside of the gels. Gel Electrophoresis, another step in the Western Blotting process, involves the transfer of the protein samples from this gel to a sensitive membrane. I've done a lot of membrane work, involving washing the membranes, adding antibodies (primary and secondary), more washing, mixing solutions, adding chemilluminecene, blotting, and exposing and developing film.

My only complaint is that it's a lot of hurry-up and waiting. However, cleaning, reading papers and books to get background information on our work, and filling in my lab book keep me busy.

All in all, I'm really looking forward to the next few weeks. I really feel that I lucked out, both by getting into the program, and getting assigned to the Kridel lab.

CERTL Program

2005-06-10T11:02:00.000-05:00

Well, I made it into the CERTL Program, as you could probably guess from the title. It's a 6-week research program with Wake Forest University and Baptist Medical Center. This summer, it runs from June 13th to July 22nd.

My faculty mentor in the Program is Steven Kridel, PhD., a member of the cancer biology department. He, along with his lab-tech Frances Wheeler, graduate student Joy Little and myself is trying to find a way to either treat or prevent cancer. Their work deals primarily with the enzyme Fatty Acid Synthase, and expanding upon the idea that by inhibiting FAS production, tumor cells themselves can be inhibited or killed.

More will surely come later as I actually start to do research with the program.