Cell theory cell fraction

1. Cell Theory

2.  In 1838, Matthias Schleiden, a German botanist, concluded that,
plants were made of cells and that the plant embryo arose from a single
cell.
 In 1839, Theodor Schwann, a German zoologist and colleague of
Schleiden’s, concluded that the cells of plants and animals are similar
structures.
 Schleiden and Schwann proposed the cell theory:
The three tenets to the cell theory are as described below:
 All organisms are composed of one or more cells.
 The cell is the structural unit of life.
 Cells arise from pre-existing cells.

3. Discovery of cells:
 The cell was first discovered by Robert Hooke in 1665.
 However, Hooke did not know their real structure or function.
 During this time microscopes having a low magnification, Hooke
had observed the empty cell walls of plant tissue.
 Hooke was unable to see that internal components to the cells.

4.  Anton van Leeuwenhoek is another scientist who saw these cells
soon after Hooke.
 He was made microscope containing improved lenses that could
magnify objects almost 300 (X) fold.
 Under these microscopes, Leeuwenhoek found motile objects.
 He states that motility is a quality of life therefore these were
living organisms.

5.  Credit for developing cell theory is usually given to two scientists:
Matthias Jakob Schleiden AND Theodor Schwann.
1. All living organisms are composed of one or more cells
2. The cell is the most basic unit of life.
 In 1855, Rudolf Virchow added the third tenet to cell theory.
3. All cells arise only from pre-existing cells

6. The modern version of the cell theory:
 The modern version of the cell theory includes the ideas that:
1. Heredity information (DNA) is passed on from cell to cell.
1. Energy flow occurs within cells.
2. All cells have the same basic chemical composition.

7. Prokaryotic and eukaryotic cells:

8.  There are two basic classes of cells prokaryotic and eukaryotic:
 The eukaryotic cells almost certainly evolved from prokaryotic
ancestors.
 Because of their common ancestry, both types of cells share an
identical genetic language.
 a common set of metabolic pathways,
 and many common structural features.
 and both types of cells are bounded by plasma membranes.

9. Prokaryotic cell Eukaryotic cell
The size is 0.1- 5.0 µm The size is 5-100 µm
Cell wall, if present, contains
peptidoglycan
Cell wall, if present, contains
cellulose, pectin and chitin.
A typical nucleus is absent. A typical nucleus made of nuclear
envelope, chromatin, nucleoplasm,
nuclear matrix and nucleoli
DNA is generally circular. DNA is commonly linear
DNA is naked or without any
association with histone
proteins.
DNA is associated with histones.

10. Prokaryotic cell Eukaryotic cell
Introns are commonly absent in
DNA, RNA, therefore, does not
require splicing.
Introns are quite common. RNA,
therefore, requires spicing
process.
Cell membrane may have
infolding called Mesosomes.
Mesosomes absent
Mitochondria's are absent Mitochondria's are present
Ribosomes are 70 S Ribosomes are 80 S
Cell organelles absent Cell organelles present

11. Prokaryotic cell Eukaryotic cell
Sexual reproduction is
absent.
Sexual reproduction is present.
It may have pili and
fimbriae.
Pili and fimbriae are absent
Transcription occurs in the
cytoplasm
Transcription occurs inside the
nucleus.
Translation occurs in the
cytoplasm
Translation in the cytoplasm

12. Cell fractionation:

13. Cell fractionation:
 Cell fractionation is the process used to separate cellular
components.
 Most cells contain a variety of different organelles, proteins and
enzymes.
 To study a particular function of organelle or protein or enzyme,
first isolate the relevant organelle or protein or enzyme in a purified
state.

14.  Isolation of a particular organelle in bulk quantity is generally
accomplished by the technique of differential centrifugation.
 In centrifugation process particles of different size and shape
travel toward the bottom of a centrifuge tube at different rates
when placed in a centrifugal field.

15.  To carry out this technique, cells are first broken by mechanical
disruption using a mechanical homogenizer.
 The homogenate is then subjected to a series of sequential
centrifugations at increasing centrifugal forces.
 Initially, the homogenate is subjected to low centrifugal forces for a
short period of time.
 so that only the largest cellular organelles, such as nuclei, cell wall
debris (and any remaining whole cells), are sediment into a pellet.

16. 1

17.  At greater centrifugal forces, relatively large cytoplasmic
organelles (mitochondria, chloroplasts, lysosomes, and
peroxisomes) can be spun out of suspension.
 In subsequent steps, microsomes and ribosomes are removed
from suspension.
 This last step requires the ultracentrifuge, which can generate
speeds of 75,000 revolutions per minute, producing forces
equivalent to 500,000 times that of gravity.

18. Purification of a protein:
 Chromatography techniques generally used for the separation of
proteins or enzymes from the homogenate.
Ion-exchange chromatography: Separate the proteins based on charge.
Gel Filtration Chromatography: Separate the proteins based on size.

19. Ion-exchange chromatography:
 Ion-exchange chromatography depends on the ionic bonding of
proteins to an inert matrix material.
 Two of the most commonly employed ion-exchange resins (inert
matrix material) are Diethylaminoethyl cellulose (DEAE) and
Carboxymethyl cellulose (CM).
 DEAE-cellulose is positively charged and therefore binds
negatively charged molecules; it is an anion (-) exchanger.
 CM-cellulose is negatively charged and acts as a cation (+)
exchanger.

20. Column

21.  The resin is packed into a column, and the protein solution is
allowed through the column in a buffer whose composition
promotes the binding of some or all of the proteins to the resin.
 Proteins are bound to the resin reversibly and can be displaced
by increasing or changing the ionic strength (or pH) of the
buffer. (which adds small ions to compete with the charged
groups of the macromolecules for sites on the resin).
 Proteins are eluted from the column in order from the least
strongly bound to the most strongly bound.

22. Gel Filtration Chromatography or size exclusion
chromatography :
 Gel filtration separates proteins (or nucleic acids) primarily on the
basis of their effective size.
 Like ion-exchange chromatography, the separation material consists
of gel beads that are packed into a column through which the
protein solution slowly passes.
 The materials used in gel filtration are composed of cross-linked
polysaccharides (agarose or Sephadex G-150 beads) of different
porosity, which allow proteins to diffuse in and out of the beads.

23. For example if a solution consists of three different proteins such as 75 kDa and
25 kDa and 120 kDa.,

24.  For example if a solution consists of three different proteins such as
120 kDa, 75 kDa and 25 kDa.
 To purify 120 kDa protein form mixture, the sample pass through a
column of Sephadex G-150 beads.
 When the protein mixture passes through the column bed, the 120
kDa protein is unable to enter the beads and remains dissolved in the
moving solvent phase.
 The gel beads allows only the entry of proteins that are less than
about 100 kDa size.

25.  As a result, the 120 kDa protein is eluted as soon as the preexisting
solvent in the column (the bed volume) has dripped out.
 In contrast, the other two proteins can diffuse into the interstices
within the beads and are retarded in their passage through the
column.
 As more and more solvent moves through the column, these proteins
move down its length and out the bottom, but they do so at different
rates.
 Among those proteins that enter the beads, smaller species are
retarded to a greater extent than larger ones.
 Consequently, the 120-kDa protein is eluted in a purified state, while
the 75-kDa and 25 kDa protein remains in the column.