Lesson 27 - Cell fractionation

Question 1

what is cell fractionation?

Answer 1

the process where cells are broken up (lysed) and the different organelles within the cells are separated out

Question 2

what must happen before cell fractionation can begin

Answer 2

the tissue is placed in a cold, buffered solution with the same water potential as the tissue

Question 3

what are the 3 conditions the solution must be and why do they have to be that condition?

Answer 3

1. Cold - to reduce enzyme activity that could break down the organelles
2. Buffered - so that the pH doesn’t fluctuate otherwise it could denature the enzymes
3. Same water potential - to prevent organelles bursting/shrinking as a result of osmotic loss or gain of water

Question 4

what are the stages of cell fractionation after the solution has been added?

Answer 4

1. Homogenization (of the tissue)- this breaks open the cells, usually done by vibrating the cells or grinding them up in a homogeniser (blender). This releases the organelles from the cells.
2. Filtration - the resulting fluid (homogenate) is filtered to remove any debris (whole cells or large bits of remaining tissue)
3. Ultracentrifugation - the fragments in the filtered homogenate are separated in a centrifuge at increasing speeds

Question 4

What is produced when centrifugation has taken place?

Answer 4

supernatant, less dense part of the cells.
pellet/sediment, more dense organelles/parts of the cell.

Question 5

what will happen when centrifugation is repeated at progressing speeds?

Answer 5

This will fractionate cell homogenates into their components

Question 6

How does cell centrifugation separate cell components?

Answer 6

Cell centrifugation separate cell components on the basis of size and density. The larger and denser components experience the greatest centrifugal force and move most rapidly. They sediment to form a pellet at the bottom of the tube, while smaller, less dense components remain in suspension above, a portion called the supernatant.

Question 7

What organelles/parts of the cell get separated into the pellet at these centrifugation speeds?
low speed
medium speed
high speed
very high speed

Answer 7

low speed - whole cells, nuclei, cytoskeletons
medium speed - mitochondria, lysosomes, peroxisomes
high speed - microsomes/other small vesicles
very high speed - ribosomes, viruses, large macromolecules