PAGE Races

Welcome to the PAGE races! I will give a quick introduction to all of our competitors before we begin our 1 hour journey through pores big and small. Ladies and gentleman please  fasten  your safety belts. We will be traveling at a comfortable 200 volts and do not anticipate losing cabin pressure, but in the event that we do....just kidding. Here are the samples and the lanes they were loaded in.
Lanes:
1. Brain (non-hematoma)
2. Brain (non-hematoma)
3. Brain (hematoma)
4. Brain (hematoma)
5. Blood (sheep heart artery/ new)
6. Blood (sheep heart artery/new)
7. Blood (sheep heart artery/old)
8. Blood (non-sheep)
9. Urine
10. Bio-Marker

And the winner is......I can't tell. The polyacrylamide gels that I am using are long enough for the tiny proteins that i am trying to extract. The protein that I am after is around 4,000 kDa. This gel only goes to 10,000 kDa. The good news is that the proteins are actually moving in the gel  which they were not doing with the previous SDS buffer I was using. This makes the smell of this new stuff worth it. The bad news is that the proteins are still a streak at the end of the plate. We have vertical electrophoresis tanks here in the lab which I can try. The only problem is that the gels cannot be purchased and polyacrylamide in it's polymer form, according to C3PO can "cause a very unpleasant death". I quickly imagined a bugs death and decided to research another way to run these vertical gels. I found a study that successfully used a 3% agarose gel (the bigger the percentage the smaller the pore size). My first attempt at pouring these gels was not successful. 1.8 grams of agarose is dissolved into 60 mL buffer solution. The solution is then injected into 2 panes of glass (about 8X11"). However, when I injected the gel it started to coagulate halfway down the glass. Oh, I almost forgot, the pieces of glass are so close together, the only thing I could find that would fit in-between them is an 18 gage needle. I also grabbed a used mini-gel cassette and super glued it together in order, and filled it with my new agarose solution to see if it can be reused. My second attempt at puring the vertical included the plates clamped with a rubber seal in-between the glass and placed in a water bath. This was also not successful due to water breaching the seal and compromising the gel. I have a new idea for attempt #3 but I will not ruin the surprise now :)
It was great to see you all in the lab today. Thank you Jeremy for all of your help this week (including all of your witty banter).
Also Thank you to Josh who has been so amazingly patient and accommodating. I appreciate the time you spend helping me pour through journal articles and combining all of the brainstorming sessions.

Electrophoresis

I am so happy to be back in the lab again! I am seeing many new faces and would like to say "welcome" to all of the new interns this semester. Words cannot describe how happy it made me to see familiar faces (Josh, Germie, Matt, Paul, D) you guys have become my friends. Now, enough sentiment...
 I am excited to continue with my project from last semester and I was super busy this week. My goal for this week was to complete a protein extraction using PAGE (Polyacrylamide Gel Electrophoresis). The most common protocol uses a steel ball mill (which we do not have and which is very expensive) so I was forced to be creative and create an alternative method (thanks Matt and Josh for the brainstorming sessions). I used sheep brains and hearts, placing 1 gram into a eppendorf tube with 250 uL of SDS buffer. I threw in a ball bearing and placed on the  vortex until pulverized.  This worked amazingly. Samples were then frozen, placed in boiling water, and centrifuged. The supernatant was extracted and a loading dye was added. Finally I set up my tank and loaded the gel. I am happy to report that the extraction was a success! However, the specific proteins that I am trying to extract have an atomic mass of 4514.04 kDa and the mini-cassette does not measure that small. Thwarted again! After another brainstorm session I have decided to perform another extraction using a vertical electrophoresis system. I had to become familiar with this contraption because I have never used one before and it looked a bit intimidating. Also, I will have to pour my own gels for this one and I was using polyacrylamide (which was pre-made and sent to the lab) which can be fatal in powder form. This didn't sound like a great idea to play with so I am going to try out a agarose gel. Monday I will make the cassettes and Tuesday I will load them and run them. Hopefully it will be a success!

New semester new protocol

It is great to be back in the lab. This semester I will continue my exploration of the Beta-Amyloid proteins and antibodies associated with Alzheimer's Disease. I was hoping to be able to obtain these proteins and/or antibodies but they are extremely expensive. The protocol that I used last semester extracted proteins from fish, but did not extract proteins from the brains or blood. So my first step was finding a protocol for protein extraction from brain tissue. Once again, the solutions needed for the extraction protocol that appeared to yield the highest results are very expensive. I have combined a few different protocols and with the help of Matt and Josh I am ready to begin this journey. My first challenge came when my protocol called for a pH 6.8 Tris HCl solution and the Trisma base  we have in the lab has a pH of around 9.8. With the help of a pH meter (which needed to be calibrated) and some HCl I achieved my first mission. Next I needed to make an SDS buffer which was simple to mix but one of the smelliest things I have ever been exposed to. Beta- mercaptoethanol is the worst smelling compound that I have ever smelled (the rest of the lab will agree, love you guys).  The next step of this protocol was to pulverize the brain tissue in a ball mill grinder (which, again is very expensive). After a brief meeting with our local lab droid (C3PO) I decided to simulate a ball mill grinder. This meant placing 81 ball bearings (from the bearings on my roller skates) into a falcon tube along with the brain tissue and 250 uL of SDS buffer. The falcon tube was placed on the vortex for 8 minutes. Next week I will be preparing samples, loading PAGE gels and performing electrophoresis to see if this smelly protocol works.

 

Presentation Week

Well the semester is coming to an end and I am not ready stop! This semester has been amazing. I learned so much working on this project. Although my final paper has been turned in I still have a few more tests that I would like to complete before this semester finally ends. I know know that proteins can be extracted from tissue samples, and that antibodies can be extracted from these proteins. I know that sheep brains can contain the beta amyloid proteins that are responsible for Alzheimers Disease (AD). Today I found a study that was performed at a university in Japan that was super empowering. They successfully measured the Beta amyloid proteins from individuals receiving treatment for AD. Now, all that is required is to put this all together and continue to work on my technique. Hopefully next semester will yield a tangible test that can be used in everyday application. However, my secondary hypothesis has proven to most challenging (Is it possible to consume an entire bag of tasty oh's in a semester?)

Just Keep Swimming!

This week has been absolutely crazy for me. I had three exams and final project deadlines are quickly approaching! This week in lab I have worked on trying to come up with an idea for obtaining brain slices that are thin enough for me to see proteins but strong enough to survive the staining process. I started by selecting three brains that are positive for frontal lobe hematoma and three brains that do not have any visible hematoma. I am not sure that there is any significance to this but, I am curious as to why approximately 40 percent of the brains in an unopened bucket are positive and the rest are not. I then used a coring instrument to obtain three samples from each brain and placed them in the -80 degree freezer for 24 hours. The coring process was not much fun, the preservative smell and the close proximity needed for an accurate sample left me feeling a bit nauseous. Perhaps I will do only three at a time for my next coring session. Next I very slowly  made slices using my makeshift microtome. I then took these slices and stained them using crystal violet. I have been able to slice them extremely thin but once I start staining them they disintegrate, littering the sink with brain matter which is a bit gross.  The problem with such thin slices is that they thaw once they hit the room temperature microscope slides. Perhaps freezing them again before staining along with the microscope slides will help? I also got to spend time discussing projects with some of my S-STEM colleagues, celebrated Matt Hills birthday, and tried to offer a warm welcome to our newest STEMmers who were busy with their unknown microbe projects. All in all, it was a great week and I am excited to see everyone's presentations in the very near future!

Hematoma Brain

Normal Brain

S-STEM Field trip

Last Friday I had the good fortune of my first S-STEM field trip. We went to Dreamy Draw park and I got to hike and experience geocaching for the very first time with some pretty amazing people. Thank you to our fearless (and extremely knowledgeable) tour guides Matt and Josh. I learned about femoral drainage and the mining of mercury that used to take place there. It was a much needed break from this stressful semester, and I am so happy that not only has S-STEM been an amazing experience, but I did not realize the bonds that would be formed from spending so much time together in the lab.

This is a pic of Matt and I attempting microtome #2

The search for a new brain

This week has been an exciting eek for me in the lab. I have successfully created a microtome! Ok, it is a very archaic design, but functional nonetheless. I used a 20 cc syringe, a brass corer (size 7), and a blade from a box cutter. The tip of the syringe was removed using a box cutter. After much measurement I realized that the diameter thickness that I wish to acieve just happens to be the distance between the cc markings on the syringe. Matt Hill used a saw to create an opening for my blade. the brain is cored and placed into the syringe. a smaller corer is used to plunge the brain down the syringe until it is pressed against the blade resting in its position within the cut in the syringe. when the blade is released the brain is moved ever so slowly forward and the blade slices through it. This little device has created some adequate samples for staining (using crystal violet) so I am very happy with it. However,  the lego microtome is still in the process of being built (thanks to Matt Hill) but is not quite finished yet.  The first brain that I sectioned was chosen due to the presence of a hematoma on the frontal lobe and a decreased frontal lobe size. This brain contained proteins when stained and viewed under the microscope. However, frozen brain tissue is much easier to cut so I placed it in the -80 degree freezer so that it would be super frozen. Well, when I applied the pressure to it in order to core it...it exploded into pieces which flew all over the bench and floor. although impressive it was really unpleasant to clean up (it defrosts quickly in such small pieces.) Now brainless, I picked another brain randomly from the bucket. after getting a sample under the microscope I quickly realized that it did not contain the proteins that I was looking for. I chose another brain randomly and again obtained negative results. I began searching the buckets for a third brain vowing to be picky this time to find one that shared the same characteristics as the first brain. I found the perfect specimen at the bottom of the bucket. Unfortunately I ended up dipping my leather banded watch in the process of retrieving it, which my fellow STEMmer Jeremy found extremely entertaining. This brain is positive for proteins. I am not sure if this is coincidence or not but it is defiantly interesting.

Beta amyloid present in brain tissue.

http://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAYQjB0&url=http%3A%2F%2Fwww.scbt.com%2Fdatasheet-53822-beta-amyloid-20-1-antibody.html&ei=Kk9cVNP1MY72iQLCn4G4AQ&bvm=bv.79189006,d.cGE&psig=AFQjCNGoJW91fjtiLzgLVGozUWqAPsl6bA&ust=1415422087904595