Isolation of Mitochondria via Differential Centrifugation

 In this blog post, I’d like to talk about a lab experiment I performed with my lab partner, Adrianna Wuerslin, in March of 2013. The experiment was performed in the cell biology lab in Hudson Hall at SUNY Plattsburgh under Doctor Donald Slish. The primary focus was on differential centrifugation. Differential centrifugation is a method used to separate organelles from whole cells based on density. The focal point was to separate mitochondria from the whole cell of a cauliflower. After two centrifugations all of the mitochondria were expected to be in pellet 2. The first was spun at 600xG and the second at 12,000xG.

 Figure: Showcases the fractions after each centrifugation.

Isolation of the mitochondria was detected by a marker enzyme. It is important to have an enzyme marker that is specific to mitochondria so the isolation can be tracked. Succinate dehydrogenase (SDH) was the marker used because it is bound in the inner membrane of the mitochondria. Being mitochondrial specific, the goal was much easier reached. 

 

By differential centrifugation and a marker enzyme, the concentration of mitochondria was found in the floret of a cauliflower. After all calculations and results were done, my partner and I concluded that we had several errors, most due to bad data. The problem can be seen in all folds of enrichment, especially S1. A fold enrichment of 3.85 is very high. High fold enrichment means that there was very little protein in that fraction. S1 should have had more protein than P1 essentially as well, but P1 also showed more amounts of protein due to a lower enrichment fold compared to S1. Because data was so skewed in the first centrifugation it makes sense that the second centrifugation results did not look well either.

  Table: Showcases the the fold enrichment in each fraction.

Column	Specific Activity (un/mg)	Fold Enrichment
H	0.11
P1	0.28	2.52
S1	0.42	3.85
P2	0.01	0.09
S2	0.08	0.72

 I believe this was a very educational experiment and would enjoy performing it again to see if I can get better data.

*All of this information (except the table) was taken from the sections (Introduction and discussion) that I solely completed in the lab report.
*The table, my lab partner, Adrianna Wuerslin, and I both completed.