Brains, Stains, and Pains

So this week was filled with good news and bad news. The bad news is that my western blot was not successful, however I did learn a ton from just completing the process. The good news is that I have completed collecting data for this part of my project and I am confident that I can extract the amyloid proteins should I be lucky enough to find a sample that contains them. I read an interesting article pertaining to these specific proteins being present in the brains of sheep. This is super lucky for me due to the abundance of them in  our lab. I have read many articles related to staining proteins and which stains work best. It seems that; crystal violet, Iodine, and comaisse blue seen to be the most popular. I prepared brain samples on slides and used each stain just to see if one was easier to interpret than the others ( and because I was super curious). I liked the crystal violet best but my samples were too thick. A microtome would make this step so much easier but they are pretty expensive (although a lego version is currently under construction thanks to Matt H and Josh).  I need each sample to be no thicker  than 250 nm. Stains have been ordered, so next week promises much fun!

This Weeks Post is Brought to you by the Letter J

Josh, J-E-L-L-O, jousting....three words that describe this week in the lab. Protein extraction through electrophoresis was not something that I was not familiar with before this semester. I successfully cut up my frozen fish samples into 0.25cm, watched C3PO add DDT to it (its harmful if inhaled), placed it in the water bath for 5 minutes at 95 degrees Celsius, loaded my gel (which comes pre-assembled in a very hard plastic cassette, added buffer to the tank, set the volts at 200, and crossed my fingers for about an hour. After the cycle was complete I rinsed the gel cassette and realized that this particular cassette requires a "key" which is not included, but can be purchased for $5.00 (plush shipping/handling). Let's Just say that I was  not pleased with the manufacturers of this particular product. I had spent hours on this particular gel and was not going to lose my results because of a silly "key". I did some quick investigating of the mechanics of this cassette and decided that the most rational approach would be to  just break it. I began by using the plastic comb that was removed from the wells of the gel but the cassette was not budging and was not giving up. Luckily Josh (the biker scout), sensing my frustration at Jousting with this cassette, came to the rescue with an extremely large flat head screwdriver. I quickly accepted this gift, had a fleeting thought of smashing the cassette with the tool, and finally decided to take his advice and use it to pry the thing apart. After a few minutes the cassette acquiesced and released my beloved gel. After it was rinsed and stained (thanks again to Josh for help with this process) I recorded my results. I was left with a gel full of data that was going to start to degrade which gave me a very sad feeling. I was not ready to part with this little masterpiece so I immediately went online to find out how others have successfully handled this situation. The answer was immediate and brilliant.....J-E-L-L-O! Gels can be preserved in a 90/10 Jello solution for long lengths of time. Now....on to Western Blotting!!

 

 

One Fish, Two Fish....

This week I started the protein extraction process from five different fish species. Biker Scout provided me with salmon and swai (which I have never heard of) and we had a lively conversation of whether or not canned tuna fish would work for this project (the answer was no, but we both decided that it would be fun to try later). C3PO came to the rescue (with the help of the lab-mobile and some mechanics provided by our biker scout) and provided very aromatic samples of catfish, dover, and halibut (which everyone else in the lab appreciated).. I am very curious to find out how successful this technique is; for I would like to try more evolved organisms after I master the fish. I also spent some time swabbing a fellow stemmer which turned out to very amusing and fun after the third hour. My fish have been cut up into 2.5 cm3 pieces and are marinating in buffer solution as I type this. My hope is that I will have results for my next post.