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!
Thursday, February 12, 2015
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!
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!
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