Gentlementlemen

Warning: This post contains music by The Cardigans ft. Tom Jones. Just go pause the video at the bottom of the post to stop it, that is if your man enough to do it.
Today is my final day of my internship.

I'll be presenting a PowerPoint that contains everything that I had worked on over the past 3 months. So that also covers my presentation I have to do for graduation. I also have my poster completed, though it still needs to be printed.

I know in my lats blog post I said that this week was going to be the biggest week yet. Well, I lied. This week was pretty normal. I counted the cells that I stained and found out that BiP is highest in areas were GLUT-1 is moderate. Which may be linked to a new biological pathway. The cisplatin treament to the tumors also worked. The tumors that were treated were much smaller than the ones that weren't.

That's all I really have to say. I haven't done anything else new since my last post.

Hmmm.

Thinking of something.

I guess I can make daily flashes. A new flash a day until I graduate or something like that. I could do that if I don't get distracted by vidya games and such. I'll need to get some awesome inspiration cause I'm pretty bored right now. I'm sorta hungry too.

Western Blots and Stuff!

Warning: This post contains Simon and Garfunkel. The video is at the bottom if you want to turn it off.

Since I have an hour to spare while my slides get incubate in antibodies I thought I would blog.

So, yesterday we finished the whole western blot process and... it was a success! Hooray~

I guess I should explain what happened.

So... we took the lysates I made, and inserted them into a gel. The  gel then goes into a vertical electrophoresis tank and runs for about two hours until the proteins have finished moving.

This is that step. Wikipedia has EVERYTHING!

This is what it looks like when the electrophoresis is done. The blue is the proteins. The heavier proteins are the ones that moved the most.

After the gel is done running, we take it out, stick a positively charged membrane on it, and sandwich it between charged plates, with currents running through them. Through the power of magic (well actually because of the charges of the plates and the proteins) the proteins move from the gel, through the 9th dimension (not really), and then onto the membrane. See below for a picture!

IS THERE ANYTHING WIKIPEDIA CAN'T DO?!

Once the proteins are on the membrane we do something called blocking. Blocking is when we have antibodies attach to the proteins we don't want to have other antibodies attach to. We do this by have the membrane rock back and forth in non-fat dry milk. Yes milk. From a cow. Moo~. After we blog we added the primary antibody which binds to the proteins that we want. Then we added a secondary antibody which attaches to the primary antibody, making it visible. No picture for this. Wikipedia has failed me.

After the all that antibody stuff was done, we placed the membrane into a film cassette with special film inside. We went into the dark room and started to develop the film. This the room was pitch black I have no idea what went on inside, but I have a feeling it involved pixies and fairies and other magical creatures. So, the cassette goes inside a machine that takes a picture a three different exposures: 30 seconds, 1 minute, and 5 minutes. After the film comes out, there are little black rectangles on it. The deeper black the rectangle is, the more proteins are in it. Well, the our controls were very dark and the proteins that came from the virus cells were very light. This means that the PERK that I mentioned was knocked down, which means that the cells are no longer making that protein, which means next week I can do an in vivo trial (I take the PERKless cells and inject them into a chicken egg then give the chicken chemo to see if the cells are weaker or not).

This what the end result looks like. I would've taken a picture of the one we did, but I don't know where my Adviser put it and she is busy right now :P

Next week is the biggest week EVER!

The staining I'm doing right now is pretty important though. This time is serious business. I took the best tumors, sliced them up, put them on slides and started staining (well duh). I'm staining for both BiP (which was explained previously) and GLUT-1 (which is a membrane bound protein that shows up a lot when the cell is under hypoxic stress). So I have stained 2 slides with BiP, 2 slides with GLUT-1, and 2 slides with BiP and GLUT-1. So when put under a flourencent microscope, the areas with BiP are green, which should be around the center of the tumor slices, while the areas with GLUT-1 are red, and should show up only around the membrane of the cells within the hypoxic regions of of the tumor slice. If everything works we should have awesome pictures of the tumor that can be published.

That's all for today since my computer is about to die any second.

Bloggle

Exciting week last week. I did some new things. Yup. New stuff. Lemme explain all of it.

We needed proteins for a western blot. 

Western blot

–noun Biology, Medicine/Medical .

a highly sensitive procedure for identifying and measuring the amount of a specific protein in a mixed extract, as in testing for aids virus protein in a blood sample: proteins are separated by gel electrophoresis and transferred to a special filter paper, on which the protein under investigation can be detected by a probe, as the binding of a labeled antibody.

 Thanks dictionary.com. I sure couldn't explain that.

Anyway, to do a western blot we needed proteins. To get proteins, we needed to murder some cells. But not ordinary cells you see. We need to harvest the special cells which I will explain now. This post is becoming an explanation within an explanation within an explanation. BUUUUUUUUUUUUUUUUUUUUUUM! (Inception reference :3)

Two weeks ago (I forgot to blog about this) I did something called transfection. Tranfection is when I take some nice little cells from our tumor cell line and jack them up with some retroviruses. But with a twist. These viruses contain a specific sequence of genes that when injected into a cell they will silence (turn off) the genes we don't want, like the genes that create PERK. So then those little cells can't make PERK anymore :( No need for dictionary.com this time. This process takes about 4 days. 1 day to set up then 3 days of incubation in 32 degrees (Celsius btw) incubators.

Back to the explanation within the explanation, we needed to get some proteins, by preparing lysates. This takes a bit, especially if your have several different sets of cells. First, I had to suck the nasty media off the cells and clean them with some PBS (phosphate buffered saline) then add some tris-EDTA (hydroxymethyl with Ethylenediaminetetraacetic acid [say that 5 times fast] acid in it) to break the calcium bonds holding the cells to the plate so I can put the cells into a comfy tube. After they're in the tube I need to count how many cells are in the tube using a hemocytometer. Here's a picture.

If looked through a microscope you'll see a grid. Count the cells in the grid and do a calculation to get an accurate amount of cells within the tube.

After counting I need to split the cells up into two different tubes. 2 million cells go into one tube while the rest go into another. The 2 million cells get spun down and frozen in a -80 freezer for storage. The other tube gets spun down and taken to the bench. This is the part where I kill them. I wash the cells again in PBS then I cover them in RIPA (Radio-Immunoprecipitation Assay which helps lyse the cells) with β-Glycerophosphate disodium salt (which stops dimerization). More spinning down the cells occurs. Blah blah blah. Lysates! Then they get stuck in the fridge until needed.

And that's it. Oh, and I'm getting a hair cut today.

Yo, ze.

So, the past week at work I've been working on florescent staining techniques. We have this anti-body that attaches to BiP (which I mentioned in a previous post) and glows neon green when a blue light is shined on it. The thing I hate about staining is the incredibly tedious work that goes into it. It is probably so easy a caveman can do it (derp) but you sorta lose your mind and screw up some how. The whole process takes about 9 hours, though it is usually split up into two days of work. The worst part though is every three freaking minutes, you have to make the slides into a different chemical or apply some kind of antibody on it. You can't go off and do something else for most of the staining process. You have to sit and wait, thinking to your self for a majority of the time. GAH! Even thinking about it makes me angry.

By the way, this is what my staining looks like. That is when it works.

Anyway, I did a total of three full stainings. The first time went okay. The problem was that the slides had too much background and non-specific staining(which is like fake green that gets in the way of the real green, so you can't tell if your looking at BiP or just some overexposed bit of cell). The second time, I totally f***ed up and added anti-bodies to the control bits of the slides (which aren't supposed to have anti-bodies on them, you know, since they're controls) and was too agressive on the slides and washed the tissues off. But the third time was perfect. All the slides had barely any background and non-specific staining, which was a huge pick-me-up for me and Aparna.

With that said, next week I take those amazing slides and take pictures for the journal and the grant and other sciency stuff.

And 2 Weeks too Late

Here's a flash to pass the time. It is science related.

I Can't Think of a Crafty Title

So last week was pretty amusing. Everything went better than expected. You see I had accidentally mixed two plasmids together, which could have completely screwed up the week. Luckily, we managed to save the plasmids from the previous vial. Crisis adverted.

Work so far has actually been pretty fun. I know right. Work, fun, the same sentence. The lab that uses the bench across from ours is pretty crazy. But in the good way. The guys there help keep the lab fun. Below is a typical day in the lab with them.



 So as I was saying about the plasmids. Since they were fine, we proceeded to transform them with our bacteria. The process involves injecting the plasmids into the bacteria. Heatshocking the bacteria. Giving the bacteria delicious media to live in. Then shaking them really fast in a bacterial shaker for an hour. After the bacteria has been shaken to perfection, the bacteria can be put into larger tubes with more food in it. But there is a trick with this part. This food is actually laced with drugs that will kill all the bacteria we don't want. Yay bacterial genocide! Those cells shake overnight. The next day the bad bacteria is dead and the bacteria master race is ready to be put into a large prep.

The culprit of bacterial eugenics

I also learned how to use the Plasmid Maxiprep System. It's just like the internet, a series of tubes.

Chinese subtitles are for clarification

So to use this wacky contraption one must first have their bacteria transformed. After a large prep of the bacteria is made the fun can begin. First the bacteria must be pelleted in a centrifuge. While keeping the pellet intact, extract the media from the bottle. Take the pellet and wash it with chemicals such as lysing solution and neutralization solution, you know that stuff under the sink your mom told you not to drink. This process will kill and remove all the cells while keeping the DNA intact. Now, as seen in the picture above, take the clear tube and attach it to the vacuum tube of the blue caterpillar vacuum device. As not seen in the picture above, take the blue tube and stack it on top of the clear tube. Pour your plasmids into each blue tube and wait until the liquid from the blue tube falls into the clear falls into the vacuum. After waiting for a long while, trash the blue tube and begin another exciting wash, cleaning the clear tubes. After your clear tubes are squeaky clean, the plasmids should have accumulated on the cotton sheet at the bottom of the clear tube.  Take the clear tube and place it into a 50mL conical. Fill the conical with 1uL of nuclease free water and spin the conical down for five minutes. Remove it from the centrifuge afterword, leaving it in there will accomplish nothing. Now hold your plasmids high in the air and feel triumphant knowing that you hopefully didn't screw up.

Ah, yes. Work.

I seriously hate blogging. The fact that I'm doing this is even surprising to myself.

Anyway, I started my work at the University of Arizona's Building of Medicine. Well, actually I started two weeks ago, but whatever.

Besides the fact that I wake up early every morning  regretting that I stayed up so late the previous night, I really enjoy working in the lab. From 9 to about 4 everyday, I do SCIENCE. Well, SCIENCE is a bit vague but I'll cover it later in this post.

My work is exactly like this (That's a lie)

So in the lab it's just me and Aparna (My advisor). I am the only person working with her. No one else. Just me. The main focus of our research is to identify hypoxic regions of the tumor and determine if BiP and/or PERK, ATF4 or Nrf2 co-localize with regions of hypoxia and also to determine whether down regulation of BiP and/or PERK enhances the sensitivity of hypoxic regions of tumors to therapy induced cell death. What this all means is that we're looking at the kinases BiP and PERK and lowering the amount of them within tumors to see if the tumors become less resistant to chemo-therapy and radiation treatments. Fun right?

First of all, we need tumors. So we order fertilized chicken eggs, open a centimeter by centimeter square in the shell and inject tumor cells into the the chicken. We then put the chicks back into the incubator and wait a week. Four days before the chicks hatch we sacrifice (Yes "sacrifice is in fact the correct term for this :3) by gently cutting off the top of the shell were the tumor was injected. Then we literally toss the fetus into the trash. Yes, I know, it's very humane.

This would be sorta what I do

Partially relevant

I'll add more to this when I get my interest back. Oh yeah and if there are spelling errors, ヽ(´ー｀)ﾉ I ain't even mad.