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.