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.