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.