I thought I would tell you about how bored I am at work today....what I am doing? A time course (TC) experiment.....not a hard one, just harvesting cells every hour, spinning them down, washing them in phosphate buffered saline (PBS), spinning again, then freezing cell pellets on dry ice (if you are a stickler you would use liquid Nitrogen)... I set up the experiment with a control treatment and a drug treatment....each to be harvested hourly.
and for what...so I can bust open the cells on another day...all samples at once collected over the TC, using detergent plus some protease inhibitors (so my proteins of interest do not get chewed up while the samples are being processed).....and then run the cell lysates on SDS-PAGE, which is very short for sodium dodecyl sulfate polyacrylimide gel electrophoresis.... and them blot the proteins which are now embedded with the SDS-PAGE gel and separated by size onto a membrane (like nitrocellulose or nylon) using a active transfer apparatus...meaning with current....so that once the protein is stuck on this membrane it stays there. This "blot" can then be probed with various antibodies on interest that are specific for interesting proteins or modifications of proteins (like phosphorylated forms).....and then these can be visualized using a secondary antibody that is tagged with a enzyme (like horse radish peroxidase) and specific for the primary antibody used...when substrate is added.....
So what am I looking for? Well, in this case I am trying to figure out the course of events that lead to cell death induction in cells treated with my compounds of interest.
At this point I am looking at proteins involved in cell cycle checkpoints, mostly with those involved in S phase/G2. The lucky candidates for now are Chk1 and Chk2 (and phosphorylated forms), and CDC25.
Anyway, you are probably all as bored as me by now....off for another timepoint....