BCH 4033L- lab 3: cell fractionation by differential centrifugation

procedure parts

homogenization

filtration

purification

• isolation of nuclei

• isolation of mitochondria

• staining of nuclei and mitochondria

clean up

what does perfuse mean?

washing the blood cells from the liver by injecting an isotonic solution into the tissue

what does homogenize mean?

breaking down cell membranes to release the intact cell organelles into a fluid medium

what is a pellet?

the particles sedimented out of a suspension, found at the bottom of the centrifuge tube

what is a supernatant?

the clean liquid layer above the pellet

what were the components of the fractionation buffer?

2 mM magnesium chloride

3 mN calcium chloride

10 mN Tris

0.25 M sucrose

what was the role of the magnesium chloride in the buffer?

it stabilized the nucleus during homogenization

maintains the nucleus’ integrity and prevents it from separating and contaminating other fractions

what was the role of the calcium chloride in the buffer?

it neutralized the charge on the biological molecules

what was the role of the sucrose in the buffer?

it was used as an osmotic stabilizer

what was the role of the Tris in the buffer?

it was the specific buffer used and resisted any change in pH (kept pH at 7.4)

how was the nuclei isolated?

the homogenate was centrifuged at 1700 rpm for 10 minutes at 4 deg. C

the supernatant was decanted and saved

fractionation buffer was added to the nuclear pellet and resuspended by vortexing

• the contents of the 2 tubes were combined

centrifuge at 1700 rpm for 10 minutes at 4 deg. C

supernatant was decanted and discarded

pellet with nuclei was resuspended in 1 mL fractionation buffer

how was the mitochondria isolated?

tube with supernatant from nuclei isolation was centrifuged at 3500 rpm for 10 minutes at 4 deg. C

the supernatant was decanted and discarded

pellet with mitochondria was resuspended in 1 mL fractionation buffer

how was the nuclei and mitochondria stained?

1 drop of resuspended nuclei or mitochondria was placed onto a glass slide

1 drop of methylene blue (for nucleus) or Janus Green B (mitochondria) was placed on drop of sample

glass cover slip was placed on solution and sat for 5 minutes so liquid could spread out

slide examined under 40X objective in a light microscope

why was the homogenization necessary?

to reduce the liver sample into small particles to prevent separation

how can a sample be homogenized?

glass/Teflon Potter-Elvehjem homogeniser

grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, ultrasound homogenization

how was the liver homogenized?

using a Glass-Teflon Potter-Elvehjem homogeniser (gentle)

done in an isotonic solution to prevent osmotic damage

done with a pH buffer to regulate pH

done at an ice-cold temperature to prevent enzyme damage

why was the sample filtered?

because animal tissue is likely to yield connective tissue that must be removed

done by pouring through cheesecloth

why was the sample purified using differential centrifugation?

to separate organelles according to their density