- What subcellular components are being separated?
- Why do we use centrifugation?
- What are the expected results?
- What are in the specific fractions?
- What does Succinate Dehydrogenase do?
- Why do we use OD260?
In this lab, we are isolating mitochondria and nuclei from liver cells by centrifugation.
Centrifugation separates subcellular particles by density, nuclei are more dense than mitochondria.
- If nuclei are more dense, do they pellet out of solution in the low speed or high speed spin?
- Where do we find them after spin one? Pellet or supernatant?
-It takes MORE speed to pellet out mitchondria because they are LESS dense!
Why are nuclei more dense?
- Because there is more DNA!
If the more dense particles pellet first, then...
-the nuclei are in P2K
-the mitochondria are in S2K
If the less dense particles pellet second, then...
-the mitochondria are in P20K
-the tiny particles are in S20K
SDH Assay
Succinate + FAD --> Fumarate + FADH2
***The reduction of FAD to FADH2 creates the color change!!!***
What does color change in SDH assay tell us is present?
-mitochondria
-if mitochondria are present, a Redox reaction is taking place and causing the blue to colorless change
So, if the expected results show that S2K SHOULD contain the mitochondria,
what color will it be?
Colorless!
But what if P2K is colorless? What does that mean? How could we fix it?
-Color change tells us that mitochondria are present! Uh oh!
-If we spun the mitochondria out in the first spin, the spin is too fast! Slow it down and try again and see if the mitochondria stay in the supernatant!
****THIS IS HUGE! Know what is present, what it means, and how you fix it!
Determining Concentration of DNA
Spec at OD260 (Optimal light absorption for DNA)
[DNA]= OD260 x 50ug/mL x df
If you cant remember dilution calculations, check out the Spec lab and the milk story!
No comments:
Post a Comment