Save The Fish!

FISH:
I decided to save the fish! I was sad to hear that nobody picked up Jenni's aquaponics project from last semester. I decided to take it on because they are the friendliest fish that I have ever seen. The water was really low and the plants were not looking very healthy. The pH is around 8 which is not healthy for the fish or the plants. The plants are happy with a pH around 7.5 and the fish will die at a pH of about 6.5 so pH 7 seems to be the happy number. I added water and waited 3 days to see if the pH would lower. It did not. Adding more fish (ammonium) will lower the pH but I was not sure how many fish to add. A second tank was set up to become a spare fish home. If it gets too acidic in the aquaponic tank they will have a safe place to go. 5 new fish were purchased and added to the spare tank. After acclimating, 2 of these fish were added to the aquaponic tank. Hopefully this will bring optimal pH to all of my new friends.

So many things

This week has truly been interesting . I found out that it is possible for a person to have two different blood types. It happens most often with twins exchanging genetic material in vitro and is commonly called chimerism. After receiving a letter from United Blood Services after a recent donation I was made aware that I may be one of these people. The letter also stated that they are not sure which blood I could be given if I ever required a transfusion because according to the test I have A and O blood. . I also am not a twin so this information was even more confusing. I decided to delve more into this and began by typing my blood. The test showed little to no agglutination in any of the serums. I called my mother to try to help me fill in the blanks. She stated that a fetus was removed through a D&C 27 months after my birth. After much research I learned that chimerism can be the result from one twin absorbing the genetic material from a nonviable fraternal twin fetus. I explained the situation and she confirmed that when I was 5 I had a minor surgery and she was told that I had a rare blood type and could not receive a transfusion if the need arises. She also confirmed that I was a very selfish child so the possibility of me absorbing a twin is highly probable. I would like to test my DNA to see if this is truly the case. I also want to mix different blood types with mine on a microscope slide in order to see if there is anyone that is compatible with me. A few brave souls volunteered this week but so far none of the RBC's got along with mine. I am currently looking for primers in order to get a final answer out of my DNA. 

PROTEINS:
With the help of Josh and his mad hot gun skills I was finally able to successfully pour an agarose vertical gel and it was really cool. I loaded to 50 mL syringes with smelly SDS agarose gel and loaded the vertical plate while Josh kept the glass warm with a heat gun. I am still trying to figure out the best way to place the gel and the proper voltage and running time for the vertical chamber before I perform an electrophoresis with this. I am also waiting on bio-markers before I run and electrophoresis with the denser PAGE gels.

It's all about the gels this week.

After reading a journal article titled SDS Agarose Gels for Analysis of Proteins I discovered that they were using mini-casettes (8x8x0.1 cm) that were loaded with agarose gel. I have previously been using polyacrylamide mini-cassette gels but the concentration was not dense enough to capture the specific proteins that I am trying to capture. I had some of these  mini-casettes in the refrigerator. I took them apart using the key (long flathead screwdriver), being very careful to only break the side seals in order to keep the seal at the bottom intact. I used liquid tape to secure the edges. 0.5 g of agarose was mixed with 50 mL of SDS on a hotplate. A 5 mL syringe was used to load gel. Unfortunately the liquid tape did not hold and the gel escaped enough to allow air bubbles into the cassette rendering it useless. 
I decided to try a horizontal gel to see what kind of results I got. SDS (yelloe in color) was used to pour this gel (whitish-clear in color) and the wells were loaded with brain,urine and blood. A bio-marker was loaded but will only register proteins >6. After 88 minutes of electrophoresis at 100 volts I noticed that the gel was changing color. The gel was changing from yellow to clear as the proteins migrated from the negative pole to the positive pole. The positive reservoir was also changing from clear to yellow. When electrophoresis began both wells were clear from the buffer solution. I cut this gel in half and stained half of it with comassie blue and half of it with fast blast. The comassie blue showed banding but again, the bio-marker was not adequate so I still can not calculate the molecular weight of these proteins. The fast blast was too dark to visualize any banding.
I became extremely frustrated and decided to attempt another vertical gel. I was pleasantly surprised to see a package from Genscript waiting for me. Inside was PAGE mini-cassete gels with the concentration needed to capture the 4 kda proteins. Next I will run the PAGE and attempt to successfully pour an agarose vertical gel.

Agarose gel stained with comassie blue

Brain storming

Agarose gel stained with fast blast

Reservoir well turns yellow

Agarose mini-cassette

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?)