Cellular Fractionation Experiment
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Centrifuge is a laboratory device that spins objects which are put inside it in a fixed rotation with very high speeds and uses centrifugal force to move the objects outward. In centrifugation process two different measurement units are used: RPM and RCF. Revolutions Per Minute (RPM) is the measurement of how many revolutions the centrifuge completes in one minute. Simply, RPM value tells the rotor's speed of spinning. The Relative Centrifugal Force (RCF) is the measurement of gravitational force that the rotation speed of the centrifuge applies on a sample an it is expressed in the unit of gravity (Difference Between RCF and RPM in Centrifugation n.d.).
With the centrifugation process, denser particles accumulate away from the rotation centre and the lighter ones towards the centre. The denser ones are known as pellet and the remaining solution is supernatant (Journal of visualized experiments: JoVE. n.d.).
Differential centrifugation is a procedure that is used to deeply analyse certain organelles by separating them from others considering their size and density. In this technique, particles with higher density travel toward the bottom of the centrifuge tube at a faster rate. (Centrifugation - Biology-Online Dictionary 2005).
During cellular fractionation, the temperature should be under control. To make centrifugation and fractionation effectively temperature must be kept around 4 degree Celsius to protect DNA and proteins from degradation. When centrifuge machine is started, the observer should not be leave there until maximum speed is reached in case of any possible problem like that machine may be stop. The other factor is choosing the most appropriate tissue. Tissues should be chosen based on their organelles which are wanted to be seen and examined at the end of the centrifugation and suspension processes.
Homogenization is a process in that cells are opened in an isotonic buffer to isolate different organelles from cells. Various types of homogenization can be applied to substances. Sonication is a way that uses the high frequency sound waves to break open cells. Detergent lysis is to use chemicals, French press breaks open cells by applying high pressure and mechanical homogenization is the way that uses a high-speed blender to break open cell.
There are two main types of centrifuge rotors: Fixed angled and swinging bucket. At fixed angled rotor pellet accumulates on the walls of the tube while it accumulates at the bottom when centrifugated with swinging bucket.
Since the centrifuge works with centrifugal force change in speed, gravitational force and frictional resistance affects the content of the pellet. Since rotor radius has an effect on RPM and RCF values, it also effects the centrifugation (Katkov and Mazur 1999).
The aim on this experiment was to obtain mitochondrial pellet through gradually cellular fractionation process with centrifugation machine. Besides, the aim was to observe how to homogenize and centrifugate samples, also the difference between different rotor types and their function.
- Fresh rat liver
- 0.25M sucrose
- 50 ml and 15 ml centrifuge tubes
- Ice bucket
- Table top and high speed centrifuges
- Maxi-pipette/ Serological Pipette
- Pasteur pipette
Preparation of sample and Homogenization:
- 10 ml of 0.25M Sucrose was poured onto 1 g rat liver that was taken from ice bucket.
- Prepared mixture was homogenized with blender, starting with low speed and increased.
- Sample was labelled.
The First Centrifugation.
- The homogenate was taken to table-top centrifuge working with 800 g (2037 RPM) force and centrifugated for 5 minutes at 4 degree Celsius.
- This centrifugation was done with the swinging-bucket model rotors whose model name was SX 4250.
- After centrifugation, supernatant was separated from pellet clearly by pouring it to another 15-ml centrifuge tube then supernatant was kept and labelled, and pellet parts were thrown away.
- In the end of the procedure we got cell debris, cell membrane and cytoskeleton.
The Second Centrifugation:
- The second centrifugation was made with a different centrifuge (J2 221) which was worked with 5000 g (5710 RPM) speed and fixed angle rotors whose model name was JA-14.
- Precision scale was used to balance the masses. The masses placed carefully and symmetrically.
- The samples were centrifugated for 15 minutes and again at 4oC.
- After centrifugation supernatant was kept and labelled. While separating supernatant from pellet it poured into a 15ml centrifuge tube from the opposite side of the pellet.
- In the end of the procedure nuclear pellet was seen.
The Third Centrifugation:
- This centrifugation step was made with the same centrifuge with the 2. centrifugation: J2-21 model Beckmann and JA14 model fixed angle rotor.
- To balance the opposite weights, sucrose was added to some empty tubes on the adaptors.
- After balance was achieved, adaptors were put oppositely in the centrifuge. The inside cover was closed.
- Sample rotated with 24.000 g (12512 RPM), for 10 minutes and the temperature was set between 4 and 10 degree Celsius.
- After centrifugation supernatant was thrown away and the pellet was kept this time. Supernatant and pellet were separated with serological pipette and three-way bulb. The pipette was hold on the opposite side of the pellet in a way that it did not touch the pellet.
- In the remaining part without supernatant, existence of mitochondrial pellet was recorded.
Resuspension of the mitochondrial pellet:
- 5 ml 0.25 M Sucrose was added onto the mitochondrial pellet.
- Maxi pipette was used to dissolve the pellet in the sucrose by pipetting up and down without touching the pellet and taking liquid part from the opposite wall of tube according to pellet.
- The mixture was stirred clearly with vortex.
- Mitochondrial suspension was obtained.
1 gram rat liver was homogenized with 10ml of 0.25M sucrose in order to separate cell parts and organelles with centrifuge properly. In each step the sample was placed on ice rather than being waited at room temperature to inhibit protein degradation. Similarly, in centrifugation part of the experiment to prevent protein deformation temperature was kept around 4oC. Masses on the adaptors were balanced with some tubes with sucrose to prevent any error resulting from asymmetrically placed masses on the adaptors. Moreover, to prevent any centrifuge related problem the centrifuge was waited to reach its maximum speed that is desired for the experiment.
The sample was homogenized successfully by changing its speed. After the first centrifugation with table top centrifuge working with 800g RCF and 2037 RPM values, where swinging bucket rotor was used, cell debris, cell membrane and cytoskeleton were seen in the pellet. The radius of the centrifuge was calculated as 17.2 cm.
The second centrifugation was done by using a fixed angle rotor at 5000 g RCF value which corresponds to 5710 RPM value. After this step, pellet was gathered not on the bottom of the tube as the first one but seen on the wall. The colour was darker on the bottom and lighter on top. Nuclear pellet was observed.
The third centrifugation was done by using fixed angle rotor at 24000g that corresponds to 12512 RPM value. After this step, supernatant and pellet were separated with serological pipette and three-way bulb. In the remaining part, mitochondrial pellet was observed. At the final step, the remaining pellet was suspended by adding sucrose and stirring the mixture gently until obtain a clear mitochondrial suspension.
The aim of this experiment was to observe not only homogenization and centrifugation processes but also how to take care of homogenizer and centrifuge. Centrifugation process was applied to the sample step by step till desired molecules were obtained. In this experiment, last step was to get mitochondrial pellet.
Since the purpose of the experiment was to obtain mitochondrial pellet, a structure that is mitochondrially rich should have been chosen. Liver cells are responsible for many metabolic activity resulting in needing high amount of energy to continue the process and finally abundance of mitochondria. Which is why the liver cells were the best samples to examine for cellular fractionation. (Caprette 2012)
Sucrose is a buffer that is isotonic at 0.25M and does not hinder enzymes' activity in animal tissues also not react chemically with organelles so prevents the mitochondria lysis during the process. Those characteristics make Sucrose one of the most useful buffers, since isotonic buffers are used to homogenize tissues appropriately. Also, the density and size of sucrose molecules make it easy to suspend pellets, balance centrifugation and even pour off it as supernatant at the end of each centrifugation. (Clayton and Shadel 2014)
All the centrifugation steps were carried out at low temperatures to adjust the heat revealed by friction force against the rotating speed of the centrifuge. Organelles were stored at -70°C to inhibit the cellular damage and inactivate the enzymes that lysis the cells.
In the second centrifugation, in the instruction manual the RPM value for JA-14 model fixed angle rotor was given as in between 5500-6000 values for 500g RCF and 13.7 cm radius. Yet, from the RCF = 1.119 X 10-5 (RPM)2 X r equation, the RPM value was calculated as 5710. In instruction manual, the RPM value, similarly, was given as 12500 since same rotor model was used. Yet according to our calculations the RPM value was 12512.
Totally pure organelle fractions cannot be obtained with differential fractionation since this method separates organelles based on their size and density. Thus, it is necessary to use another method that separate samples considering their density. This method is density- gradient centrifugation. It should be centrifugate samples at high speeds and for hours to let each cellular component to migrate their equilibrium positions (Lodish et al. 2000).
Caprette, R. David. 2012. "Tissue Fractionation." http://www.ruf.rice.edu/~bioslabs/methods/fractionation/fractionation.html (March 14, 2017).
"Centrifugation - Biology-Online Dictionary." 2005. http://www.biology-online.org/dictionary/Centrifugation (March 10, 2017).
Clayton, D. A., and G. S. Shadel. 2014. "Isolation of Mitochondria from Cells and Tissues." Cold Spring Harbor Protocols 2014(10): pdb.top074542-top074542. http://www.cshprotocols.org/cgi/doi/10.1101/pdb.top074542 (March 14, 2017).
"Difference Between RCF and RPM in Centrifugation." http://www.westlab.com.au/page/97/Difference_Between_RCF_and_RPM_in_Centrifugation.html (March 10, 2017).
Journal of Visualized Experimentsâ€¯: JoVE. https://www.jove.com/science-education/5019/an-introduction-to-the-centrifuge (March 10, 2017).
Katkov, Igor I., and Peter Mazur. 1999. "Factors Affecting Yield and Survival of Cells When Suspensions Are Subjected to Centrifugation." Cell Biochemistry and Biophysics 31(3): 231-45. http://www.ncbi.nlm.nih.gov/pubmed/10736749 (March 15, 2017).
Lodish, Harvey et al. 2000. "Purification of Cells and Their Parts." https://www.ncbi.nlm.nih.gov/books/NBK21492/ (March 15, 2017).
Calculation of radius of the first centrifuge: RCF = 1.119 X 10-5 (RPM)2 X r
800 g = 1.119 X 10-5 (2037)2 X r
r = 17.2 cm
Calculation of RPM values: RCF = 1.119 X 10-5 (RPM)2 X r
No.2 centrifuge: 5000 g = 1.119 X 10-5 (RPM)2 X 13.7
RPM = 5710
No.3 centrifuge: 24000 g = 1.119 X 10-5 (RPM)2 X 13.7
RPM = 12512
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