A Level Biology Revision "Cell Fractionation"
Summary
TLDRThis video from Free Science explains cell fractionation, a technique for studying cellular organelles. It starts with homogenizing tissue to break cells open, using a buffer to maintain pH and prevent organelle damage. The homogenate is then centrifuged at increasing speeds to separate organelles by size: nuclei, mitochondria, lysosomes, and ribosomes. The process requires keeping samples cool and acknowledges challenges in achieving complete separation.
Takeaways
- 🔬 **Cell Fractionation Importance**: Cell fractionation is a technique crucial for studying the functions of different cell organelles.
- 🧪 **Tissue Homogenization**: The process begins with homogenizing tissue to break it up and open cells, which can be done using a blender or a homogenizer.
- 🧊 **Use of Buffer Solution**: A buffer solution is used to maintain pH and prevent organelles from bursting due to osmotic water movement.
- ❄️ **Cooling the Sample**: Keeping the homogenizer on ice slows down enzymes, protecting the organelles from damage.
- 🔄 **Breaking Cells**: The plunger in the homogenizer is used to disrupt the tissue and break open cells, creating a cell homogenate.
- 🔽 **Organelle Sizes**: The relative sizes of organelles play a key role in their separation, with larger ones like the nucleus being easier to separate.
- 🌀 **Centrifugation**: A centrifuge is used to separate organelles by spinning the homogenate at different speeds, causing them to settle at different rates.
- 🧲 **Pellet Formation**: Larger organelles form a pellet at the bottom of the tube after centrifugation, while smaller ones remain in the supernatant.
- 🔁 **Sequential Spins**: Multiple spins at increasing speeds are required to separate organelles of different sizes, from nuclei to ribosomes.
- ⏳ **Time and Temperature**: Keeping pellets on ice is crucial to prevent enzymatic damage, and complete separation of organelles is challenging.
Q & A
What is the purpose of cell fractionation?
-Cell fractionation is a technique used by scientists to separate cell organelles so that they can study their functions individually.
What is homogenization in the context of cell biology?
-Homogenization is the process of breaking up tissue and breaking open cells to create a cell homogenate, which contains all the organelles found in the cell.
Why is it important to maintain a constant pH during homogenization?
-Maintaining a constant pH is important because changes in pH could cause enzymes within the cell organelles to denature, which would affect their function.
What is the role of the buffer solution in homogenization?
-The buffer solution maintains a constant pH and has the same water potential as inside the cell, preventing water from moving into the organelles by osmosis and causing them to burst.
How does cooling the sample during homogenization help protect the organelles?
-Cooling the sample slows down the activity of enzymes, which helps prevent any destructive enzymes from damaging the organelles during the homogenization process.
What equipment is typically used to homogenize tissue samples?
-A homogenizer, which is a glass tube containing a plunger, is commonly used to homogenize tissue samples.
How does a centrifuge aid in the separation of organelles during cell fractionation?
-A centrifuge spins the sample, and the organelles are flung towards the bottom of the tube by the forces generated. Larger organelles move faster than smaller ones, allowing for separation by size.
What is the first organelle to be pelleted during the centrifugation process?
-The first organelle to be pelleted during the centrifugation process is the nucleus, as it is the largest organelle.
Why is it difficult to separate organelles fully during cell fractionation?
-It is difficult to separate organelles fully because some organelles of different sizes may have similar densities, and the process may not be able to differentiate them completely.
Why do we need to keep the pellets on ice after they are separated?
-Keeping the pellets on ice slows down enzyme activity, which helps prevent damage to the organelles before they are used for further study.
What other organelles might be present in the fractions besides the ones mentioned in the script?
-Other organelles such as the endoplasmic reticulum and Golgi apparatus might be present in the fractions, although they are not the primary focus of this script.
Outlines
🔬 Introduction to Cell Fractionation
This paragraph introduces the process of cell fractionation, which is a technique used to separate cell organelles. It begins by explaining the importance of understanding the functions of various organelles like mitochondria, lysosomes, ribosomes, and the nucleus. The video then describes the first step in cell fractionation, which is homogenization. Homogenization involves breaking up tissue and cells to create a cell homogenate, using either a blender or a homogenizer. A homogenizer is a specialized tool consisting of a glass tube and a plunger. The tissue sample is placed in the tube, covered with a buffer solution to maintain pH and prevent osmosis, and then homogenized on ice to slow down enzymatic activity. The resulting cell homogenate contains all the organelles, which are then separated by size using a centrifuge.
Mindmap
Keywords
💡Homogenization
💡Cell Fractionation
💡Organelles
💡Centrifuge
💡Buffer Solution
💡pH
💡Nucleus
💡Mitochondria
💡Lysosomes
💡Ribosomes
💡Supernatant
Highlights
Introduction to cell fractionation technique
Importance of studying cell organelles
Homogenization process explained
Use of a blender or homogenizer for tissue disruption
Role of buffer solution in maintaining pH and preventing organelle damage
Centrifugation as a method for organelle separation
Relative sizes of organelles and their separation
Nucleus as the largest organelle
Mitochondria's position in organelle size hierarchy
Lysosomes' size and separation
Ribosomes as the smallest organelles
Challenges in separating endoplasmic reticulum
Fractionation process using a centrifuge
Low-speed spin for initial organelle separation
Formation of pellet and supernatant after centrifugation
Higher speed spins for further organelle separation
Final high-speed spin to separate ribosomes
Testing each fraction to understand organelle functions
Challenges in fully separating organelles
Presence of other organelles in fractions
Importance of keeping pellets on ice
Transcripts
[Music]
hi and welcome back to free science
lessons by the end of this video you
should be able to describe how
homogenization and self-factionation can
be used to separate cell organelles
now cells contain a number of different
organelles including mitochondria
lysosomes ribosomes and the nucleus and
in later videos we look at the functions
of all the organelles
in this video we're going to look at a
really important technique that allows
scientists to study the functions of
organelles this is called cell
fractionation
in the first stage we take a sample of
tissue containing the cells that we're
interested in for example heart muscle
tissue
next we homogenize the tissue
homogenized means to break up the tissue
and break open the cells
now we can do this in a blender or we
can use a homogenizer like this
a homogenizer is a glass tube containing
a plunger
we place our tissue sample into the
glass tube and we cover this with a
buffer solution
buffers keep the pH constant
now this is important because if the pH
changes enzymes in the cells organelles
could denature
the water potential of the buffer is the
same as inside the cell this prevents
water from moving into the organelles by
osmosis and causing them to burst
the homogenizer is then placed on ice
cooling the sample means that enzymes
work more slowly preventing any
destructive enzymes from damaging the
organelles
now we push the plunger up and down to
disrupt the tissue and break open the
cells this produces a cell homogenite
the cell homogenite contains all the
organelles that we find in the cell
now in order to find out what these
organelles do we need to separate them
and in order to understand this we need
to look at the relative sizes of the
different organelles
the largest organelle is the nucleus
followed by the mitochondria
lysosomes are smaller and ribosomes are
very small indeed
now I should point out that the
endoplasmic reticulum is a very large
organelle but this tends to get broken
up during homogenization so we're not
going to consider this
separating out all of the different
organelles is called fractionation and
we carry this out using machine called a
centrifuge I'm showing you a picture of
a centrifuge here
we place our tubes containing the cell
homogenate into the sample holder
so here are sample tubes containing the
cell homogenite
the centrifuge now spins the sample and
the organelles are flung towards the
bottom of the tube by the forces
generated large organelles such as the
nucleus experience a greater force and
move towards the bottom of the tube
faster than smaller organelles
first we start with a relatively low
speed spin at the end of the spin the
tube looks like this
as we said as the centrifuge spins the
larger organelles such as the nuclei are
flung to the bottom of the tube forming
a pellet
the remaining organelles stay suspended
in the liquid and we call this liquid
the supernatant
we now transfer the supernatant into a
new tube uncentrifuge this at a higher
speed
after the higher speed spin the pellet
now contains mitochondria
once again we transfer the supernatant
to a new tube on centrifuge again at a
higher speed this time the pellet
contains lysosomes
finally we take the supernatant one more
time and transfer this to another tube
for a final very high speed spin
now the palette contains ribosomes
so as you can see we've separated all
the organelles by size
at this point we can test each fraction
to determine how the organelles work
now there are a couple of final points I
want to make first we need to keep the
pellets on Ice until we use them again
this is to slow down enzymes which might
damage the organelles
secondly it's extremely difficult to
separate the organelles fully
so for example the mitochondrial
fraction might contain a very small
number of nuclei and lysosomes
also there are other organelles such as
the endoplasmic reticulum on Golgi
apparatus which might be present in your
fractions
okay so hopefully now you can describe
homogenization and cell fractionation
[Music]
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