Third Update: Exercise and PGC1a Expression

Weeks and weeks of Western blots and FINALLY I was able to get a signal for PGC1a! I honestly cannot tell you how exciting it was when I realized that the dark red band was right around the target area for the gene, and I was able to quantify it as well!

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The first time I got a signal was with a mouse heart I homogenized—the concentration of the protein was very high so I was only using tiny amounts of the it in the blot. To get a more accurate measure, I homogenized a new mouse heart in twice as much lysis buffer, then ended up diluting that one again to get a concentration of about 6.74 ug/ul. This meant I was able to do a more broad serial dilution, using 10ug, 20, 40, and 60. Because the mouse heart was so large, I had enough protein to do repeated measures as well. After quantifying the bands, I graphed protein expression (band intensity) versus amount of protein. As expected, there is a linear relationship, which indicates that I did not use too high a concentration/oversaturate the membrane.

graph

 

 

Now that I have a solid signal for PGC1a, I have started moving on to our rat tissues. This past week I did a serial dilution western of rat femoral artery, which is very similar in structure to the rat aorta, as both are large, elastic arteries, though the femoral artery is a lot smaller. I used this blot to determine the optimal amount of rat aorta protein I need to get the best expression for PGC1a (~50ug).

fem art

I have moved on now to the rat aorta tissues and hope to wrap up my research this week with some exciting new insight to how PGC1a is expressed in the exercise aorta versus the controls. Finally, my project will culminate in a poster detailing the work I’ve done this summer and the results of my research. Stay tuned for my final post!

 

-Kayla