Historical Introduction

In the early 1700s, Leeuwenhoek noted that avian and amphibian blood cells contained a “clear area” almost certainly corresponding to the structure we now know as the nucleus. This represents probably the earliest recognition that eukaryotic cells are not simply sacs of protoplasm, but instead contain subcellularstructures. Although the concept of the nucleated cell became Þrmly established in the 18th and 19th centuries, the real beginning of subcellularfractionation had to await the emergence in the mid-1940s of the art form known as electronmicroscopy .

The electronmicroscope revealed much more complexity than Leeuwenhoek could have imagined. Concurrent with improvements in the techniques of electron microscopy was the development of methodologies for subcellularfractionation. Through the 1950s, these parallel approaches resulted in the discovery and isolation of the major organelles that comprise the eukaryotic cell. Finally, as biochemical functions were associated with speciÞc subcellular compartments, a much clearer picture of the eukaryotic cell began to emerge, and with it the Þeld of modern cell biology.

What is cell fractionation

• Biologists need to study certain organelles from a cell (the mitochondria of a human cell or the chloroplasts of a plant cell, for example . Isolating these organelles involves a variety of procedures collectively called cell fractionation. As a method for studying processes within organelles, cell fractionation has both advantages and disadvantages.

• Cell fractionation is where a cell are broken up and its components and organelles are separated so that scientist can observe them in isolated form .

• It can also be defined as : the separation of homogeneous sets , usually organelles, from a larger population of cells.

What we’ve learned so far using this technique :

1.Mechanism of protein synthesis

2.DNA replication and transcription

3.RNA splicing

4.Muscle contraction

5.Microtubule assembly

6.Vesicular transport in the secretory pathway

7.Importance of mitachondria and chloroplasts

in energy interconversions .

Cell fractionation methods

Involve the homogenization or destruction

of cell boundaries by different mechanical or

chemical procedures, followed by the separation

of the subcellular fractions according to mass,

surface, and specific gravity

Steps of subcellular fractionation

1 . Homogenization

2 . Differential centrifugation

3 . Further separation and purification by density gradient centrifugation

4 . Collection of fractions

5 . Analysis of fractions

1 . Homogenization

First the cells must be broken open. A variety of different methods are available; the method chosen depends on the type of experiment and the type of sample (bacterial culture or mammalian tissue sample, for example) Detergents like SDS or Triton X disrupt the cell membrane so the contents can flow out. Subjecting the cells to ultrasound waves or sonicating them will also break them open, as will agitation in the presence of metal or glass beads. Blenders may work with tissue samples but will not work with bacteria or other microorganisms.

Homogenization or Cell Disruption

Chemical : alkali, organic solvents, detergents

Enzymatic : lysozyme , chitinase

Physical : osmotic shock, freeze/thaw

Mechanical : sonication , homogenization, French press

Chemical Disruption

• Detergents such as Trition X-100 or NP40 can permeabilize cells by solubilizingmembranes.

• Detergents can be expensive, denature proteins, and must be removed after disruption

Sonication

A sonicator can be immersed directly into a cell suspension. The sonicator is vibrated and high frequency sound waves disrupt cells.

Homogenization

• Cells are placed in a closed vessel (usually glass). A tight fitting plunger is inserted and rotated with a downward force. Cells are disrupted as they pass between the plunger and vessel wall.

2 . Differential centrifugation

Centrifugation is the process of isolating components of a cell. There are two common centrifugation techniques for separating bacteria components.

What is Centrifugation

Differential Centrifugation

Differential centrifugation is the process where a homogenate (soup of tissue and cells) undergoes repeat centrifugations and increasing centrifugal force. Centrifugations is the use of increased gravity to quicken the precipitation of substances tot the bottom. The tool used here is the centrifuge, "merry-go-round for test tubes" that spin at various speeds.

The centrifuge separates the cell's parts into pellet and supernatant. The pellet are the large cell structures that are settled at the test tube's bottom. The supernatant are smaller parts of the cell suspending in liquid, the supernatant is decanted and undergoes another centrifugation. The process is repeated and increases speed with each trial to collect successively smaller parts of a cell in pellets.

• These are both test tubes attached to a centrifuge. The first picture is of a test tube that has undergone homogenization but is about to undergo centrifugation . The second picture are the results of the centrifugation and portrays the settling of large cell parts.

The preparative ultracentrifuge

• The preparative ultracentrifuge. Sample is contained in tubes that are inserted into a ring of cylindrical holes in a metal rotor. Rapid rotation of the rotor generates enormous centrifugal forces, which cause particles in the sample to sediment. The vacuum reduces friction, preventing heating of the rotor and allowing the refrigeration system to maintain the sample at 4°C.

When a centrifugal force is applied to an aqueous mixture, components of larger size and density will sediment faster

Low speed centrifugation is used to separate intact cells from medium

High speed centrifugation can be used to separate subcellular components

Tow types of centrifuge are there

Fixed-Angle Centrifugation

Swinging-Arm Centrifugation

Method of Differential Centrifugation:

• 1. Cut tissue in an ice-cold isotonic buffer. It is cold to stop enzyme reactions, isotonic to stop osmosis and a buffer to stop pH changes.

• 2. Grind tissue in a blender to break open cells.

• 3. Filter to remove insoluble tissue

• 4. Centrifuge filtrate at low speeds ( 1000 X g for 10mins )

• This pellets the nuclei as this is the densest organelle

• 5. Centrifuge at medium speeds ( 10 000 x g for 30 mins )

• This pellets mitchondria which are the second densest organelle

• 6. Centrifuge at high speeds ( 100 000 x g for 30 mins)

• This pellets ER, golgi apparatus and other membrane fragments

• 7 Centrifuge at very high speeds ( 300 000 x g for 3hrs)

• This pellets ribosomes

Buoyant density centrifugation

The buoyant density centrifugation involves viruses with densities of 1.1-1.2 g/cm and a sucrose gradient. The cell suspension is added to the top of the sucrose gradient. In this centrifugation the densest components move fastest down the tube and stops at the sucrose density equal to its own. The sucrose gradient bands at the bottom contain cell components with high buoyant densities and the components at the top have low buoyant densities.

An illustration of the sucrose gradient and the buoyant density centrifugation.

4 . Collection of fractions

Collecting Fractions-keeping samples pure

and intact

1.By hand: puncture sidewall of centrifuge tube with needle and withdraw fractions through syringe

2.Machine: gradient uploader; introduces very dense, non-miscible medium into bottom of tube, pushes fractions up to be collected from top

3.If no pellet, can collect fractions through hole in bottom of tube

5 . Analysis of fractions

Analysis of fractions-need to identify and quantify thepurified fractions, so that they can be used successfullyin downstream applicationsMethods:1.Light or electron microscopy2.Biochemical-determine presence of marker enzymes3.Assay for a protein marker with an antibody (western)4.Determine the protein concentration by using aspectrophotometer, e.g. Bradford assay5.Determine specific activity (the ratio of activity ofthe enzyme of interest to the protein concentration

How is cell fractionation used in cell biology?

• Scientists use this tool to increase their knowledge of organelle functions. To be able to do so they isolate organelles into pure groups, such as isolating the mitochondria or the nucleus.For example, by centrifugation a specific cell fraction was determined to have enzymes that function in cellular respiration. This unknown cell fraction was rich in mitochondria . Therefore there researchers obtained evidence that helped determine mitochondria were the site of cellular respiration.

Investigating Cell Function

• Differential Centrifugation allows us to look at each organelle within the cell

• We can look at the individual organelles and study them in detail

• This helps to determine each organelles function within the cell

Uses

• Separation of enzymes, hormones, RNA-DNA hybrids, ribosomal subunits, subcellularorganelles, for the analysis of size distribution of samples of polysomes and lipoprotein fractions.

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