Wednesday, July 27, 2016

Western Blot

Today went really great, I was able to conduct a western blot using gelatin and an electrical supply. Western blots tell you the different levels of proteins in different parts of the cells nucleus.

I entered the lab and I was going to do a gel run. Essentially, you have these slides and you put nitrocellulose that has been washed with rabbit and goat protein onto the slide. Nitrocellulose is a material that is actually made from pure cellulose. Pure cellulose is what makes up cotton. It can be put into a paper-like form by a process in which the nitrocellulose is dissolved in a solvent. Nitrocellulose was what used to be used to make movie film and various plastics. Although it was of great use, it was soon found that it was far too flammable for practical use. I have made it many times, and on ignition it always flashes and makes a satisfying “whoompf” noise that never ceases to bring a smile to my face. The proteins are washed onto the slide and what is interesting is that these proteins actually help the cell samples to cling to the nitrocellulose and stain it. Think of it sort of like a chain link. You have rabbit protein which clings to goat proteins, and then the goat proteins cling to the cancer cell proteins that are in the nucleus. There is this slide, and it has these little wells, you fill the wells with your cancer cell samples. The slide is filled with electro-conductive gelatin. The protein samples stain and make these bands at the different points depending on the weight of the protein. You also have what is called a molecular weight. The molecular weight is essentially a control. The molecular weight always falls onto one band. This way depending on the density of the band, you can tell how much of a certain protein was active and formulated inside the cell, this shows in better detail how the cell reacted to the drug and antibody. I will post some pictures in the next week’s post of the western blot results as it is hard to explain in just words.

Again, comments are very welcome and I hope you enjoyed.
Thank you for your time,

Aaron J.

Friday, July 8, 2016

Data Crunching

This week, follow me as I plot my data! I know that doesn’t sound very exciting, but I promise it might be exciting.

Unknown-1.jpeg I went to the lab this morning but there was a note on the door. “Aaron we are in a meeting so please make yourself at home in the meantime ~Choh”. So I did just that, I went in and made myself at home, I setup my laptop where I normally sit and waited. A few minutes later Dr. Helman as well as all the other researchers came down from the hallway. They explained that each week, the new “lab meeting day” will be on Thursday at 10:15 a.m. So I made a note of that and went on and started plotting my data. Plotting data is really easy once you get the hang of it. I made a table on Microsoft Excel and was able to calculate the average as well as the standard deviation of the data sets. I did another protein assay and left it in the incubator. Then I went to eat. It was interesting having to deal with the new version of excel. Mr. Choh was really frustrated with some of the newer design changes to the program. 


After that it was about time to wrap things up. It was a pretty simple day, I didn’t do a whole lot, but that’s okay. On the way back, I actually managed to ride the metro back to Bethesda station all on my own. I don’t like to toot my own horn but that was quite an accomplishment for myself.

Thanks for reading! Comments are welcome and I’ll be sure to respond.
Aaron J.

Drug and Antibody Combination

Drug and Antibody Combination

Hello Harbour Friends! This week was very exciting, I looked at the results from the previous weeks plates where I administered drug and antibody therapy to the cells.

First off I have 2 new cell lines. I will test these cells and share the results next week. On my plate, all the wells grew fine, except for a single well, it was contaminated with nasty fungus. We checked each well under a microscope and what was surprising, was that the fungus killed most of the cancer cells! Of course, this would not make effective therapy, as it will also kill just as many normal cells, but it was still an interesting result. I ate lunch and came back to organize my notes. I had a bunch of sticky notes stuck to a page, but Mr. Choh says that if I went into a laboratory as a student with these notes that the professor would not even let me in the lab. So I spent a good hour putting all these sticky notes down on my lab book in pen and pencil.  I’ll post images of my newly organized notebook soon.


After this, I did an MMT assay, it is hard to explain, but it is basically a chemical that reacts in each well, based on how many cells have metabolic activity. I’ll post a picture to give you guys a better idea of what it looks like. MMT assay basically has a chemical that gets reduced within the cell via hydrogen bond. The beginning chemical is called 3-{4,5-demethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide which is also known as MTT. I don’t expect anyone to be able to pronounce this because even with my knowledge of chemistry, I can barely manage to stumble through pronouncing these words myself. The mitochondrial reductase changes this to {E,Z}-5-{4,5-dimethylthiazol-2-yl}-1,3-diphenylformazan which is also known as Formazan. I just call it Formazan. 

The Bromide ion breaks off and the nitrogen bonds change as the bond at the 4 position breaks its bond with the nitrogen at the 6 position, the nitrogen at 6 binds to a hydrogen. This hydrogen changes the markers chemical composition which changes its color, this acts as the indicator as seen above. 

Thanks for reading, again, comments are welcome and I’ll be sure to respond. 
Thank you, Aaron J.