A Level Biology Revision "Cell Fractionation"

Summary

This 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

00:00

🔬 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.

Keywords

💡Homogenization

Homogenization is the process of breaking up a tissue and breaking open the cells within it. In the context of the video, homogenization is the first step in cell fractionation, where a tissue sample is treated to create a uniform mixture of its cellular components. This is achieved using a blender or a homogenizer, which physically disrupts the cells to release their organelles. The video emphasizes the importance of maintaining a constant pH and preventing osmotic damage during homogenization.

💡Cell Fractionation

Cell fractionation is a technique used to separate different organelles within a cell based on their size and density. The video explains that after homogenization, the mixture is subjected to a series of centrifugation steps at increasing speeds to isolate organelles such as the nucleus, mitochondria, lysosomes, and ribosomes. This method is crucial for studying the individual functions of these cellular components.

💡Organelles

Organelles are specialized structures within cells that perform specific functions. The video mentions several organelles, including mitochondria, lysosomes, ribosomes, and the nucleus. Understanding the separation of these organelles through fractionation is key to the video's theme of studying cellular functions.

💡Centrifuge

A centrifuge is a machine that spins samples at high speeds to separate substances based on their densities. In the video, it is used to perform cell fractionation by applying different centrifugation speeds to separate organelles according to their size and weight. The centrifuge plays a central role in the process of isolating organelles for further study.

💡Buffer Solution

A buffer solution is used to maintain a constant pH during the homogenization process. The video explains that covering the tissue sample with a buffer solution is important to prevent enzymes from denaturing due to pH changes. It also ensures that the water potential of the buffer is the same as inside the cell, preventing organelles from bursting due to osmotic pressure.

💡pH

pH is a measure of acidity or alkalinity and is critical in biological processes. The video stresses the importance of keeping the pH constant during homogenization to prevent enzymes within the organelles from denaturing, which could compromise the integrity of the cellular components being studied.

💡Nucleus

The nucleus is described as the largest organelle in the cell and is the first to be separated during fractionation due to its size. The video uses the nucleus as an example to illustrate the principle of separating organelles based on size and density.

💡Mitochondria

Mitochondria are organelles that are involved in energy production within the cell. The video mentions that mitochondria are separated from the homogenate after the first low-speed spin, indicating their intermediate size and density compared to other organelles.

💡Lysosomes

Lysosomes are smaller organelles that contain enzymes for breaking down waste and cellular debris. The video explains that lysosomes are separated from the supernatant after a higher-speed spin, showing their smaller size compared to the nucleus and mitochondria.

💡Ribosomes

Ribosomes are very small organelles responsible for protein synthesis. The video describes ribosomes as being separated in the final, very high-speed spin, indicating that they are the smallest organelles to be isolated in the fractionation process.

💡Supernatant

The supernatant is the liquid remaining after centrifugation, which contains organelles that have not yet been pelleted. The video describes how the supernatant is transferred to new tubes for further centrifugation at higher speeds to isolate smaller organelles like lysosomes and ribosomes.

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]