Rest of the week of June 6th - In the lab

I have been remiss in updating my blog....been busy :-) Also need to make sure I don't mention anything I am not supposed to since things have not been published so excuse some lack of detail.

The week dealt with learning about and observing the use of the microscope based on optical tweezers. Yes I know it's a Wikipedia reference but it had plenty of good info and some nice pictures. Of course, I don't understand some of the equations so need to talk to Mr. S. Wiley at Science Academy to explain them to me. The reason for this microscope is that it can determine the force being applied so you can actually determine how much force the motor proteins are applying. Newton's 3rd Law! As described in the wikipedia article, proteins are too small so a bead can be attached to them so that can be used for the reference point. Dr. Diehl gave me a book to look over which describes the mechanics for the motor proteins, Mechanics of Motor Proteins and the Cytoskeleton by Jonathon Howard. Besides the microscope info, I spent the rest of the week making a given protein. Will give the basic steps of what I did.....sorta like this

I started with bacterial cell stock from -80 degree C refrigerator, using a metal loop, streaked a culture plate, which contains antibiotics, and used serial dilutions to spread out bacteria. Allowed to incubate overnight, antibiotics will kill anything that does not contain DNA that we don't want. Picked a small culture, to insure only one type of DNA, to reduce possibility of mutations and place in culture test tube, allowed to incubate. Split resulting culture into two 1 liter erlenmeyer flasks, allowed to incubate in heated shaker. Both test tube and flask culturing, allows us to get a larger population work with. When solutions have reached an approximate optical density of 1.0, checked with an instrument, the bacteria is induced to make protein by adding a given reagent which tells the bacteria to start making a given protein encoded in their DNA and incubated; this is referred to protein expression. Solutions are centrifuged to harvest the bacteria, which makes a pellet at the bottom. Now the protein is inside the bacteria, but we want it, so now we lyse the bacteria. A reagent is added that makes the bacteria burst open so proteins and other stuff is released. Yuck! Again, solution is centrifuged but now it's the liquid that we want since it contains the protein we want. The liquid is mixed with a Ni NTA mixture since the protein we are interested in is attracted to it; this is used for purification. This mixture is allowed to incubate and then run through a column. The protein stays in the column and everything else goes right through. It is washed then a reagent is added so the Ni NTA releases the protein and we can recover it. It is placed in a falcon tube to be used later. Done!