Western Round 2
Who knew the military loved to party so much. This morning I walked into the lab only to be told I would be attending the Navy’s Hospital Corpsman Birthday! (I think they turned 131 or something) Anyways, there was lots of delicious cake and solemn ceremony and a good time was had by all.
In my last post, I mentioned that I had gotten a very weak signal for my western blot. After discussing it with my PI and a couple of post-docs, it was decided that I either didn’t have enough buffy coat (leukocytes), which make up less than 1% of blood by volume or I had hemolyzed the erythrocytes and made most of my white blood cells sink because they were now heavier. To overcome these challenges, I decided to try two different techniques. The first was to “snap” freeze fresh blood immediately after spinning in our centrifuge at 3000rmp for 15min and removing the plasma layer. The idea was that I could then use a scalpel and razor blade to scrap off the top layer of white blood cells leaving them relatively intact and free of any contamination from the erythrocytes. However, scraping this precious layer off proved much easier said than done because, despite being stored in a -80C freezer, it melted after about 15sec. The second technique yielded a much bigger and cleaner layer of white blood cells. In order to effectively separate the white blood cells from the red ones, I used a “Serum Separator Tube” which is essentially a vacutainer filled with some kind of goo that weighs less than red blood cells and more than plasma and white blood cells. The buffy coat layer was a little thicker than I expected (think slightly thicker than jello) and it remains to be seen whether it will yield a stronger signal than I got last weekend. However, while a strong signal would be very encouraging and show that the western is working properly, the samples I will be using for the GR ELISA have already been frozen and I don’t think it would be possible to transfer them to Serum Separator Tubes without hemolyzing them so I might have to come up with yet another technique that would allow me to extract the buffy coat relatively uncontaminated with red blood cells from frozen epi-tubes.
In other news, I finished my Lab Animal Medicine training so am now officially allowed to take sample from mice if I run out of human donors (I think I’ve stuck everybody at least once who works here) and I have begun to set up my own bench space where I will run the GR ELISA once it arrives and I have a reliable technique to extract buffy coat from the samples.