Lab Report: Cellular Fractionation

Hello everybody!

How are you? :)

In this week, I'm really busy in the lab and because of that, I couldn't write what we've done in the lab, yet. Yesterday, we made Nuclear Fractionation to some Bacteria to use their proteins  and today, to separate the proteins by using Western Blot Technique! It is a very long process and to follow truly, I read an article about the technique. At the weekend, maybe I can share a post about the Nuclear Fractionation Protocol that we used and an other post about Western Blotting :)

However, because I cannot write these posts before the whole process ends, today I will share my lab report about cellular fractionation by using homogenization technique. I think, it can be useful to read before reading the Nuclear Fractionation Protocol :)

Here it is :)

INTRODUCTION:

Centrifuge is a laboratory machine which is used for separating and isolating different substances from each other by using a motor which is able to spin the substances which are in liquid phase, at high speed. Differential Centrifugation is also one of the techniques for separating certain organelles from the cells by using their size and density differences.

Centrifuge machines have different types of rotors such as swinging bucket and fixed angle rotors. Swinging-bucket rotors can swing the sample tubes into a horizontal plane during the centrifugation process. On the contrary, fixed-angle rotors have a particular angle to fix the tubes during the centrifugation.To explain the properties of centrifuge machines, there are RPM and RCF values. RPM (Revolution per Minute) value shows the speed of the revolution that the rotor of the machine can reach and it is independent of the size of the rotor. However, RCF ( Relative Centrifugal Force) is the value of the force which is exerted on the samples in the rotor as a result of the revolution of the rotor and its value depends on the size of the rotor.

At the end of the centrifugation process, the substances in the tube separate from each other according to their sizes and densities. The smaller and less dense components move to the top of the tube and are called “supernatant”, and the larger and more dense components move to the bottom and are called “pellet”. During the cellular fractionation, densities and sizes of the needed components and environmental factors such as temperature and pressure have to be considered to obtain true and utilizable components from the sample.

Homogenization is a process in which the plasma membranes of the cells are ruptured and the cells are broken open. As a result, all the contents of the cells can be released. During this process, the cells are placed in an isotonic buffer and the resulting mixture is called “homogenate” which contains different, large and small components of the cells such as organelles, metabolites and enzymes. To homogenate cells and tissues, high frequency techniques(sonication), high pressure to make the cells move through a small hole(French press) and techniques to shear the cells(mechanical) can be used to break the cell membranes and soft detergents(detergent lysis) can be used to make holes on the cell membranes.

The centrifugation process and its results are affected by different properties such as RCF value, the duration of centrifugation, the shapes, sizes and densities of the cell samples, the density and the viscosity of the medium solution and, the column of suspension’s length. It is needed to be careful about the balance of the samples in the rotors and the lids of the tubes and the centrifuge have to be closed carefully and, until the centrifuge reaches the maximum speed, the samples shouldn’t be left.

AIM:

The aim of the experiment was to obtain mitochondria of the liver cells by using centrifugation technique.

METHODS:

1.      Homogenization: 

·         From an ice bucket, 1gr rat liver in a 50ml centrifuge tube was taken.

·         10ml of 0.25M sucrose solution was added in the tube.

·         By using homogenizer, a colloidal mixture of the rat liver and sucrose solution was prepared.

·         The homogenizer was used until there wasn’t any apparent liver piece in the tube.

2.      First Centrifugation:

·         A Table Top Centrifuge (swinging bucket) with RCF = 800g and RPM= 2037 values was used in the first centrifugation.

·         The centrifuge was arranged before the experiment to 4^oC.

·         The 50ml falcon with 10ml mixture of liver and sucrose solution was placed in the rotor of centrifuge.

·         To balance the rotor during the process, the samples in the falcons were placed just opposite sides of each other.

·         The centrifuge was activated.

·         The machine was waited until it reached the maximum speed.

·         After 5 minutes, the sample was taken from the machine and it was observed that the pallet was at the bottom and the supernatant was at the top of the falcon.

·         The supernatant part of the sample was poured into a 15ml centrifuge tube to be used in the second centrifugation.

·         The pallet wasn’t used.

3.      Second Centrifugation:

·         To centrifuge at a higher speed, a High-Speed Centrifuge (fixed-angle) was used.

·         To determine the true RPM value that corresponds the needed RCF value, the instruction manual of the centrifuge was used.

·         To get the RCF = 5000g value, RPM = 5500 was calibrated on the machine.

·         To prepare a proper 15ml falcon with sucrose solution for balancing the sample in the rotor, the scales was used.

·         The centrifuge was arranged before the experiment to 4^oC.

·         The adaptors with falcons were placed in the centrifuge and the lid was closed.

·         The centrifuge was activated.

·         The machine was waited until it reached the maximum speed.

·         After 15 minutes, the sample was taken from the machine and it was observed that the pallet was placed on the wall of the tube.

·         The supernatant was poured into a new 15ml falcon without taking the pellet to be used in the third centrifugation

·         The pallet wasn’t used.

4.      Third Centrifugation:

·         To centrifuge at a high speed, a High-Speed Centrifuge (fixed-angle) was used.

·         To determine the true RPM value that corresponds the needed RCF value, the instruction manual of the centrifuge was used.

·         To get the RCF = 24000g value, RPM = 12500 was calibrated on the machine.

·         To prepare a proper 15ml falcon with sucrose solution for balancing the sample in the rotor, the scales was used.

·         The centrifuge was arranged before the experiment to 4^oC.

·         The adaptors with falcons were placed in the centrifuge and the lid was closed.

·         The centrifuge was activated.

·         The machine was waited until it reached the maximum speed.

·         After 15 minutes, the sample was taken from the machine and it was observed that the pallet was placed on the wall of the tube.

·         The supernatant was taken by using 5ml serological maxipipette without touching the pellet.

·         The supernatant wasn’t used.

·         Into the falcon with the pellet, 5ml of 0.25M sucrose solution was added.

·         The vertex was used to resuspend the mitochondrial pallet.

 Mitochondrial suspension was obtained.

DISCUSION:

The aim of this experiment was to obtain mitochondria of the liver cells for using them in different researches by using centrifugation technique. For this purpose, firstly the liver tissue was homogenized in 10ml of 0.25M sucrose solution. After that, three different centrifugation processes were performed and at the end of the experiment, the mitochondria of liver cells were obtained as the pellet.

For cellular fractionation, liver tissue was chosen because liver cells have more mitochondria than other tissues. Because of their work in the body, these cells require more energy than other cells and as a result, in their cytoplasm there are a lot of mitochondria to produce energy.

To perform the centrifugation processes truly, a proper solution for the sample was used. 0.25M sucrose solution was chosen to be used for this experiment, because it is an isotonic and dense solution. In addition to that, sucrose is a covalent molecule and it doesn’t dissolve ionically in a solution. On the other hand, sucrose molecules have a resolving effect for the other molecules in the sample and as a result they help to separate the compounds of sample, too.

All steps of this experiment were carried out in relatively lower temperatures because, the proteins and protein based structures in the liver cells and their components such as organelles and enzymes could be affected by high temperatures. As a result, these proteins and protein based structures could be denaturated and nonutilizable in the room temperature. Because of that, the liver tissue was kept in an ice bucket and the centrifuges were optimized at 4^oC during the centrifugation processes.

On the other hand, to determine the true RPM value for the centrifuge, the instruction manual was used. The instruction manual shows the true RPM value for the needed RCF value and it was for RCF=24000, RPM = 12500. After the centrifugation, by using the formula to convert the RCF value to RPM value, the needed RPM value was calculated mathematically, too. The result was almost the same = 12552.

By using cellular fractionation, it is not possible to obtain a complete pure organelle solution. Because this method based on size and density differences and during the centrifugation process, the compounds of the cells could not be separated accurately, especially in this short time interval. Because of that, to obtain purer results the methods which can target the needed component directly can be used, such as antibodies and magnetic beads to separate the components.

I hope, you've had a perfect and productive week and you will have an amazing weekend at the end :)

See you soon..

LOVE YOU <3

Kumsal