July 27th - In the Lab

This week I need to start working on my poster for next Monday's poster presentation where all the IBB HHMI/NSF students and teachers will show their projects.

Working on another part of animation...Heating spatula and streaking plate...took some thinking...

LINK

Starting to put things together in animation...LINK

JUST FOUND OUT THAT IF YOU ARE NOT ON A FAST CONNECTION THE ANIMATION MAY NOT WORK. I need to add a preloader or something like that. It's always something :-) You can still Rt. Click and Save as onto your desktop and then double click, when it asks you what program to use, browse and select whatever your browser your using

July 20th - In the Lab

Have been working with making more "of a given" protein

Also, continuing some work on the Flash animation. Really think the Biology teachers could use it. Still a work in progress... LINK

Quick update to the 17th....

Will update the rest of the week later....

Figured I would create an animation to explain one of the things I am doing and can be used by the biology teaches back home. It is in progress, but I got this far: LINK

July 13th - In the Lab

Back in the lab..... See what I will do this week...... Also this week, IBB Symposium, flyer, schedule....

Also, on Saturday, I will be attending the Texas College and Career Readiness Symposium on Chemistry at UNT-Dallas, just for fun, come on its chemistry :-). Seriously though, I need to go especially to find out what students are expected to know when they get to college. Will share what I can.

Rest of the week was spent working on optical trap microscope. Trying out different experiments to see what procedures should be used in analyzing motor proteins in two situations: in vitro vs. in vivo. I noticed something interesting in the Diehl lab, and I'm sure it happens in other labs. The students not only require "simple" lab skills for using glassware and other lab equipment. They are chemists, biologists, software programmers, setting up program for LabView for optical trap microscope, engineers, building a fluorescence microscope, and bioengineers, making a given gene for experiments.

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!

July 6th - In the Lab

After a couple of days off for vacation, hope everyone had a Happy 4th, its time to get back to the lab......

Time flew by.....

Been studying the chemistry behind the whole thing I am working with. I thought I was just reading not really picking anything up. A student asked me a question on what I was doing, online, and I was able to describe quite a bit :-) I said so much that it surprised me how much I actually picked up and understood. I must apologize to Dr. Hassan Ahmad, one of my professors at UTPA, since I never took his Biochemistry course because I figured I would not like it since it had the prefix bio-. I can see now how interesting Biochem can be based on my readings on how the motor cells use the chemical energy in GTP or ATP. Simple animation to see this. Exciting stuff!

July 2nd - In the Lab

Back in the lab......Will write later what we did...... :-)

I looked up more info on motor proteins and, consequently, the cytoskeleton. Read up on actin filaments, intermediate filaments and microtubules which are part of the cytoskeleton. The microtubules are the ones we are interested in since the motor proteins "walk" along them. Microtubules have a plus and minus side. Plus side is where polymerization of tubulin occurs and the minus side is unstable so it must be attached to centrosome. Kinesin motor proteins move towards the plus side; endoplasmic reticulum seems to be moved by the kinesin. Dynein motor proteins move towards the minus side; Golgi apparatus seems to be moved by the dynein. Taxol is an important chemical when dealing with microtubules since it binds to them and stabilizes them.

July 1st - In the Lab

More research.... I really don't know any Bio :-( But I am learning now :-) More about motor proteins and ......

I spent most of the day getting information so I can understand what we are doing a little better. I first looked up how motor proteins move using ATP. I found lots of information in a book titled Molecular Biology of the Cell (By Bruce Alberts, John Wilson, Alexander Johnson, Hunt, Julian Lewis, Martin Raff, Keith Roberts) It provided me with a wealth of information on motor proteins and microtubules. It explained how it was the hydrolysis of the ATP by the motor proteins that provides the energy. It also explained the differences between the three different family types of motor proteins: kinesin, myosin and dynein (the one we are working with). I won't bombard you with lots of info; let's just say it was enlightening. :-) Click HERE if you want to see a sample of the information

June 30th - Dr. Diehl's Lab

Quite a bit of work done....Need to check what I can and can't write here. Some stuff could still not be out and I am not supposed to mention those things. Top secret, if I tell you, I would need to ....... :-)

We started off the day with well blended bovine brains in buffer solution. Brains must be cleaned of protective tissue and as many blood vessels as possible (luckily Jonathan had done this already). The mixture is centrifuged and the supernatant (the liquid on top) is collected since it contains the microtubules and motor proteins. A solution is added supernatant to polymerize the microtubules (which will contain motor proteins) and placed in warm water to incubate. Afterwards, it was slowly pipetted onto a sucrose solution, due to difference in densities, the polymerized microtubules sink below the sucrose. Once set up, it is centrifuged and pellet of microtubules at bottom is collected and washed with original buffer solution along with some polymerizing agent and then incubated and centrifuged again and pellet is collected. Pellet is then suspended in buffer, ...... , and ATP solution. The ATP provides energy for the motor proteins to separate from microtubules. Solution is incubated at 37 degrees C, which is about body temperature, when motor proteins are more active. Solution is centrifuged and separated into nine, 100 microliter samples. Eight of the samples are frozen in liquid nitrogen and one is going through analysis. The sample went through some more procedures (secret stuff :-) ) and then analyzed using a certain type of microscope. (again there are some things I cannot mention, so please don't ask.....)

June 29th - Dr. Diehl's Lab

Back in the lab today. Will mention later what I worked on.......

Will be working for a few days with Jonathan, so I can learn some basics.

But first read the article from PCCP that I mentioned last time and will read some basic info on expression of recombinant proteins in E. Coli. Learning some biochemistry!

From The QIAexpressionist 06/2003