Friday, June 29, 2012

Week 6-Seeking the Bigger Picture & Meeting the Team

After hearing about a variety of research projects that are underway and have been completed on this campus, I am now even more excited about continuing my ongoing project on heart disease. With the legitimate concerns about pandemics of AIDS and behavioral disorders, it's easy to forget that cardiovascular disease kills more people each year worldwide than any other disease. We've grown so accustomed to hearing about heart disease that we even consider it a natural cause of death and tend to disregard a comprehensive solution to an obvious growing issue. I can even attest to the effects of heart disease, as it has personally plagued loved ones and family friends. Some of the most recent well-publicized deaths are even linked to such: Whitney Houston suffered from atherosclerosis, a disease of the arteries as a result of fatty materials such as cholesterol. The best part about joining this lab and its ongoing project is the fact that I feel like I am tackling two developments at the same time--discovering the role of a particular protein in heart disease and finding a solution to modulate this protein and, perhaps, a solution to heart disease in general through pharmacodynamics. I am ecstatic that this isn't just a project that stops at the discovery of a particular thing--it seeks to improve the human condition through application.

Even more exciting is the fact that one of my research team members, Ashley, is on the brink of submitting a huge paper that includes data/data analysis dating back (whew..a mouthful) to 2010! A lot of what she is including in her paper predates the contributions I have made since joining the lab; however, the implications of a successful publication are enormous for the rest of the lab work to follow.

Speaking of my research team, allow me to introduce you to some of them! They have been a part of Dr. McConnell's lab for as long as he has been a PI, and I can assure you they are a TREMENDOUS help (and motivation) to my research experience:

Name: Cori Wijaya
Education: B.S. in Biochemistry-University of Houston
What sparked her interest in researchShe started doing research work during one summer in her undergraduate senior year. She found that doing hands-on work in the lab helped her with her studying: what she read in her textbooks actually started to make sense! As she graduated and started working in the lab, she found it exciting to do experiments that helped to elucidate the questions and hypothesis of the research work.
The best part about her experience in Dr. McConnell's lab: Getting to know people who share her knowledge and skills
Advice she gave me about continuing to pursue research: Keep a curious and open mind. Research is a long process with so many little things to take care of-but be glad that you are a part of it. Spend a long enough time in one lab to learn something, but also give yourself a chance to try a different lab and see how other labs work differently.

Name: Abeer Rababa'h
EducationMaster degree in Clinical Pharmacology/ Jordan University of Science and Technology
What sparked her interest in researchShe has an interest in correlating and interpreting data into biological facts that help downstream in achieving new clinical aspects.
The best part about her experience in Dr. McConnell's labResearch with Dr. McConnell gives her the opportunity to interpret many findings in a logical way, find explanations for every single piece of data, convert bored data into exciting findings, look for something from a different point of view in a comfortable friendly research environment.
Advice she gave me about continuing to pursue research: My advice is to get as much experience as you can from the lab. Every small contribution may participate in a big achievement in your career goal. Challenge yourself and be patient. Being hard working is a requirement for the research road.

Name: Andrea Diaz Diaz
EducationB.S. in Biology and Biochemistry-University of Houston
What sparked her interest in research: She took some classes in college with a wonderful teacher that motivated her to do research.
The best part about her experience in Dr. McConnell's lab: She gets to do surgeries (on mice, that is!)
Advice she gave me about continuing to pursue research: It is a very interesting career where you can learn many things about any topic you are interested in!

X-Ray Film Processor

Developing the Film-Western Blot

Labeling the Film

Discovery Workshop-Using EndNote for your References

Program: EndNote

Heart Tissue Stored in Ethanol

Collected Heart Tissue

Moving Tissue to Histology Cassettes

Moving Tissue to Histology Cassettes

Friday, June 22, 2012

Week 5-Western Blots Galore!

My primary focus this week was continuing the development of various Western Blots. One of the members of our research team, Abeer, came back this week from maternity leave to continue her project. The lab was especially busy this week because a lot of buffer solutions had to be prepared in order to practice the cardiomyocyte isolations that will begin next week.

One thing I really enjoy about this research experience is learning (and practicing) various biological lab techniques that was only briefly mentioned in my biology class. Before entering the lab, I had only heard of a "Southern blot." This technique is used to detect a specific DNA sequence in DNA samples and uses probe hybridization. Even though the names are similar, a Western blot is completely different. It targets the identification of specific proteins in a given sample through gel electrophoresis and antibody hybridization.

The first step in a Western blot is to prepare the tissue using a sonicator (there are various techniques in preparing the tissue, but the primary one used in the lab is with a sonicator). The sonicator breaks down the solid cell wall of the cells in the particular sample and enables the protein found within the cell to be freed.

The next step is to perform gel electrophoresis. This separates the proteins of the sample by electric charge and molecular weight. Smaller proteins move faster through the gel and the proteins are separated by size. The proteins move through a specific agar gel; the more concentrated the agar gel is, the better the resolution of the lower molecular weight proteins. (agar gel is created using a powder, diluent, and heat - similar to jello!) Conversely, the lower the concentration, the better the resolution of higher molecular weight proteins. The samples are loaded into walls in the gel, with one lane reserved for a marker. This marker is a commercially made mixture of proteins having pre-determined molecular weights. They are also stained to produce highly visible and colored bands.

Running Buffer
Samples to be Loaded

Loading Samples

Loading Samples

Electrophoresis with an Electric Current

After running the gel, the proteins are transferred to what is called a membrane (PVDF paper). The primary method for transferring the proteins to the membrane is a process known as electroblotting.  This uses an electric current to to pull proteins from the gel into the PVDF membrane. What I find really unique about this process is that the electric current pulls the proteins from the gel onto the membrane in the exact same order and size as on the gel!  As a result of this process, the proteins are exposed on a surface and this is used for detection.

Next, steps must be taken to prevent the antibody from binding to a non-target protein. This technique is called blocking and it involves using TBST (Tris-Buffered Saline with Tween 20). The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. When the antibody is added, there is no room for it to attach other than on the binding sites of the specific target protein.

Detection may be perhaps the most important step in the entire process and is when antibody hybridization occurs.  In my lab, we practice this in two steps: a primary and secondary antibody. The PVDF membrane is probed with a particular antibody (mouse antibody, rabbit antibody, donkey antibody, etc.). When a match occurs, a different color on the membrane is produced.

Last, the membrane is incubated with ECL (a solution used in chemiluminescence) which allows for chemiluminescent detection. The light is detected by a photographic film and results are printed.

Other activities this week included autoclaving glass bottles to fill them with buffers and practicing cardiomyocyte isolations in order to adequately perform next week's activities.

Autoclave Unit

Autoclaved Bottles 
Cardiomyocyte Isolation

Friday, June 15, 2012

Week 4-Dealing with Collected Tissue

After tons of paper-writing, peer editing, and data-analyzing from the previous week, this week required much more activity. Some of the mice from our experimental groups finally reached 21 weeks of age and were ready to get their blood pressure & heart values confirmed by an invasive procedure dealing with the carotid artery. With this procedure completed, it was time to carefully stain the collected aortas in order to conduct lipid lesion formation analysis.

First, I drained the aortas with a 100% propylene glycol solution. This dehydrates the tissue and prepares them for staining. Next, I incubated the aortas with the stain for 4 hours at room temperature. After the 4 hours were completed, I washed the aortas with 85% propylene glycol solution. Last, I incubated the aortas overnight with a PBS (saline) solution in order to preserve the tissue and prevent it from drying out.
Collected Tissue 
The Staining Process 
100% Propylene Glycol

Collected Tissue

PBS Solution
In addition to this process, I learned how to filter the substrates to make the stain solution! This required a biosafety cabinet and a fume hood to catch the vapors of this potentially hazardous substance. This process also requires the use of a vacuum to help filter the substrate from the liquid. With a special device that connects to the top of the bottle, the vacuum acts on the substrate found in the upper portion of the device and drains all of the liquid to the bottom jar where it is then collected and stored. Because the stain is light-sensitive, we made sure to cover the bottle in aluminum foil to prevent the light from changing the chemical properties of the stain.

Filtering Oil Red O

Oil Red O Solution-Final Product

Other activities this week included washing the membranes in ongoing Western Blots, switching out the PBS solution that was used to store the collected aortas, prepration of TBST & milk, and storage of membranes from Western Blots into sealed packs.
Washing the Membranes 
Incubating Western Blot

Membrane from Western Blot

Friday, June 8, 2012

Week 3-A Very Happy Birthday and Some pH-un!

This week came to prove that a phenomenal research team and a great work environment really do make an opportunity like this worthwhile. Aside from observing and performing lab experiments, there's a lot of thinking and collaborating. Every person on the research team is examining something different, but at the end of the day, everybody's individual parts are brought together to answer a broader question. Additionally, while research may require a lot of patience and thinking, there is definitely a "fun" dimension involved.

In the process of blogging about my team's specific project details, it's necessary to be wary of what I post. This is because if too much information is publically posted about a particular experiment or our overall research goal, someone else may come to identify what our research project is about, and perhaps, publish it as their own. To avoid this, I must do two things: 1) Avoid detailing the particulars of what my research team and I are investigating before official publication 2) Avoid posting media of our specific procedure on a given specimen. I wish to share more with you, but unfortunately, until publication, I am limited to what EXACTLY I can say. Just know that alongside my lab protocols and techniques I perform, there's a lot more going on that I can't reveal right now. :)

One of the members of my lovely research team had her birthday this week-Andrea! Just because we are also working intensely to meet deadlines and submit papers doesn't mean we can't sit back and have a little bit of fun! To celebrate her birthday, one of the other members of the team, Cori, bought a delicious cake and brought it in. Every person on the research team, including Dr. McConnell, came in to celebrate and sing a version of "Happy Birthday" in their respective languages. After we celebrated, the inevitable discussion of what needs to be done in the lab and problems some of us are encountering in meeting particular deadlines ensued. Even though I have officially been a member of the lab for less than 3 weeks, I already feel like I've been here for years. It's definitely an enchanting feeling.
The Birthday Cake!

Happy Birthday!
The Research Team! (missing a few)
Furthermore, I continued to make more TBST solution in the lab. TBST is probably one of the most commonly used solutions in our research lab because we are dealing with a high volume of Western blots.  I also learned how to make a dilute solution of an antimicrobial to place in our cell incubator. It requires 5 mL of a blue substrate called AquaClean and 1000 mL (or 1 L) of distilled water. This may sound easy, but there has to be a lot of precision to how solutions are mixed and what type of "distilled water" is used. There's double distilled water, distilled water, and tap water in the lab. Some solutions require double distilled water (commonly labeled as ddH2O) while others require only distilled water (commonly abbreviated as DW). The pipetting procedure also is quite fastidious--one must spend a certain amount of time re-filling the pipette and emptying it out before all the substrate is removed. The solution must also be mixed thorougly in order to reach proper homogeneity.

AquaClean Substrate

Cell Incubator
With all of this TBST solution-making, it's not surprising that I also ran out of the 10x TBS, the substrate used to make the TBST solution. This was a rather lengthy and tedious process, especially since it requires the use of a pH meter and careful pipetting. First, I added 24.2 g of Tris base and 80 g of NaCl to a plastic beaker. Then, I stirred the contents after adding 800 mL of ddH2O . Because TBS is a buffer, I also had to ensure that the pH of was adjusted to a certain level--7.6 In order to do this, I had to standardize the pH electrode with 3 different pH level solutions (4, 7, and 10). I adjusted the pH of my solution by adding HCl to lower the pH and by adding NaOH to raise the pH until I reached 7.6 This took a while because one drop could really make a big difference in what the pH meter read, and I did not want to go over or under the level needed. Last, I added the remaining ddH2O to the solution to make 1 L total.

Tris Base
Sodium Chloride
pH Standards

Standardizing the pH meter

Adding HCl and NaOH to adjust the pH reading

Final pH-7.6

The Final Product-10x TBS!
This week was also very unique because I experienced my first team meeting! All of the research members and our PI, Dr. McConnell, assembled in a conference room to discuss the progress of our individual projects/experiments. Since there are many deadlines to meet, the tone of the conversation was tense and hasty. Dr. McConnell expressed the necessity to identify specific substrates in one of the members of my research team's experiment. This is extremely important because in order for a research paper to be published, it needs to answer a question with very specific results. If not, then the paper may not be of value to even publish. A general answer or a general piece of evidence is not sufficient enough for publication. Furthermore, we talked about what other experiments needed to be performed while also being mindful of the fact that these experiments must be replicated several times in order to yield a significant conclusion. Although my research team seemed a bit stressed out with what seemed to be a conversation of never-ending experiments and things to do, I promised to help out as much as I could. Dr. McConnell even reminded me to help out with several of the projects so that these looming deadlines could be met.

Part of the Meeting 
DNA Gel Electrophoresis 

Liquid Nitrogen (used to instantly freeze collected tissue for storage)