protein

Co-IPs are a highly specialized approach to identifying in vivo protein:protein interactions. The protein preparation is fairly similar to a total protein prep, however, a different buffer is used for lysis, and a column in used to run the fraction through. Our lab uses micro-max columns which consist of thousands individual iron atoms fused to a primary antibody (typically C-myc or HA). Running the intial fraction through this column will grab the input, or protein complex that you are initially probing, in other words, one of the two anticipated interactors. This fraction is quantified/normalized to other samples that are later run out via SDS-PAGE. This is then transferred to a charged membrane that can be probed with a different specific antibodies, which would detect the secondary interacting protein. It is crucial to rinse or wash the membrane between probing steps to eliminate any risidual or background primary antibody that has unspecifically bound to the membrane. 

protein

Protein protein interactions in vivo are a tough phenomenon to capture. However, there are special ways to identify proteins that are capable of interacting in vivo. One approach is a method called co-immunoprecipitation (Co-IP). This is an extension off of the simplistic approach of western blotting, or, immunoblotting. This technique takes advantage of biology at its core. Scientist have developed a method to engineer antibodies that are capable to bind to whatever target, or antigen that is desired. The process begins by injecting the target protein into a host (rabbit, goat, donkey, etc…), which then generates antibodies that bind to the “foreign” protein. The animal is then bled, and syrum is isolated, and the specific protein (primary antibody) is purified. The secondary antibody is synthesized in a specific manner, where it targets the “species-specific” portion of the primary antibody (anti-rat, goat secondary). Once the appropriate / specific antibodies have been made, immunoblotting can ensue. 

round II

Before dinner, the family drank a couple of beers, and this alone is enough to stir inspiration up in this family. Shortly there after, we decided to go to the community recreational center to lay a couple of games of basketball. I was not prepared. I borrowed a pair of gym shorts from my cousin (which was okay), but shoes were my biggest issue. I had to wear semi-formal dress shoes to the gym. Can you see the potential problem at hand? We are a fairly competitive family, naturally, so I played hard throughout the first three games. At the end of this game, I decided that my feet were in trouble. I sat down and checked out my boats. Sure enough, I could tell I had developed blisters during the second game, what I did not know was the severity of the blisters. My socks were blood stained, as well as the soles of my shoes. Despite this set back, Thanksgiving was awesome.

round I

Last weekend I had Thanksgiving weekend in the Dallas area with my Dad’s side of the family. It is a serious advantage having Thanksgiving practice one week before the real deal. This allows for some stomach-stretching to occur. This is analogous to practicing for any sporting event. This time we had two turkeys, one was smoked by a BBQ restaurant in the Dallas area, Dickie’s, and the other was smoked at my uncle’s house by the man himself. It is a toss up as to which bird I preffered, but since I’m an original kind of guy, I’m obviously going to pick the turkey that was prepared in my uncle’s house. Honestly, both were equally fulfilling. The dressings were something out of this world. Both were pan-baked dressings, thick in nature, thick enough to stay attached to an inverted spoon. Cranberry sauce was NOT out of the can. The sweet aroma of the simmering cranberries filled the house. 

bakn a chkn

After my hour and 15 minute timer expires, I remove the lid and bake for another half hour or so (depending on the size of the chicken, of course). This secondary baking strategy allows the skin to crisp up a little bit and allows the internal temperature of the chicken to reach the appropriate temperature of 165 degrees. I allow the chicken to cool without a lid for about half an hour before I start carving. After all of the meat has been removed from the carcass, I will often times boil the skeletal remains to produce hearty chicken broth that I will use at a later date. In the case of last week, I make about 10 cups of chicken broth, froze 5 cups, and proceeded to use 5 cups for the base of my chicken soup, a culinary zeal rushed over me, there is not much else I can say.

bakn a chkn

I had myself a nice, relaxing weekend. Mostly this occurred as an absence of OU fans invading my town. Sunday was especially relaxing, as this was the day when I baked a whole chicken. In order to bake a chicken properly, there are a couple of ground rules that need to be followed. First, you must clean the chicken. I typically do this under cold water. During this “cleaning” process, I will take the giblets out of the body cavity, and set them aside. Next, I will pat down the outside of the chicken with paper towels. After the skin has dried a bit, I drizzle a fair amount of extra virgin olive oil on the skin and spread it around with my hands. I then rub kosher salt into the skin of the chicken. After these preparation steps have been taken, it is time to start baking. I cover the casserole dish with a glass lid and bake at 350 degrees for about an hour and 15 minutes.

okay

I just made a serious noob move in the lab, fortunately it was for the best and was overlooked as the mistake that I thought it was. Here is how it went down. One of our mutants lines in the lab is resistant to a herbicide called BASTA. Most of the plant expression vectors that I use are also BASTA resistant. Is the mistake clear yet? I cloned my insert into this plant expression vector with the identical resistant marker as the plant that I was planning on dropping the clone into. The problem is that I would never be able to select for positive transformant if both resistance markers are the same. When spraying with BASTA, I would only get positive mutant plants back, I would not be informaed that I got a mutant plant with a positively inserted transgene. This expression clone is fine for selection of transgenic plant in wild-type plants, which I negligibly forgot we were doing..