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(1) Two buffer boxes contain the buffer used in the process.
Supporting
Sample Electric field Buffer
media
The sample
Charge
Size
Shape
The electric field
Current
Voltage
Resistance
Heat
The Buffer
Composition
Concentration
PH
The Supporting media
Adsorption
Electro-osmosis
Molecular sieving
Types of ELECTROPHORESIS
Zone electrophoresis
Disc electrophoresis
Capillary electrophorsis
Microship electrophorsis
GEL ELECTROPHORESIS
What is a gel?
Gel is a cross linked polymer whose composition and
porosity is chosen based on the specific weight and
porosity of the target molecules.
Types of Gel:
Agarose gel.
Polyacrylamide gel.
• Gel electrophoresis uses a cross-linked polymers
(agarose) that contain various pores.
• Pores allow molecular sieving, where molecules e.g.
DNA, can be separated based upon there mobility
through the gel.
A highly purified uncharged polysaccharide derived
from agar.
Used to separate macromolecules such as nucleic
acids, large proteins and protein complexes.
It is prepared by dissolving 0.5% agarose in boiling
water and allowing it to cool to 40°C.
It is fragile because of the formation of weak
hydrogen bonds and hydrophobic bonds.
Used to separate most proteins and small
oligonucleotides because of the presence of small
pores.
Detection
1. Dye e.g. ethidium bromide
2. Audioradiography 32P,
3. Blotting (see later)
Uses
1. Analytical- Can determine size of DNA fragment,
2. Preparative – Can identify a specific fragment
based on size
Silver staining is usually
10-100 times more
sensitive than Coomassie
Blue staining, but it is
more complicated.
Faint but still visible
bands on this gel contain
less than 0.5 ng of protein!
Electrophoretic method that separates
proteins according to the iso-electric points
Is ideal for seperation of amphoteric
substances
Seperation is achieved by applying a
potential difference across a gel that
contain a pH gradient
Isoelectric focusing requires solid support
such as agarose gel and polyacrylamide gel
Separates proteins by their
isoelectric points (pI)
Each protein has own pI =
pH at which the protein has
equal amount of positive and
negative charges (the net
charge is zero)
IEF 4-6.5 pH
gradient
Zavialov
A.
Mixtures of ampholytes, small
amphoteric molecules with high
buffering capacity near their pI, are
used to generate the pH gradient.
Positively and negatively charged
proteins move to – and +,
respectively, until they reach pI.
PI of proteins can be theoretically
predicted. Therefore, IEF can also
be used for protein identification.
Using specific probes that are labelled specific sequences of
DNA can be identified.
There are three main hybridization techniques which vary in
the sample blotted and the probes used;
1. Northern Blot-Transfer of an RNA sample separated and
identified using DNA or RNA probes.
2. Southern Blot-Transfer of an DNA sample separated and
identified using DNA or RNA probes.
3. Western Blot- Transfer of an Protein sample separated and
identified typically using an antibody.
Blotting – Transfer of DNA, RNA or Proteins,
typically from a electrophoresis gel to a membrane
e.g. nitrocellulose. This membrane can then be
subject to further techniques such as hybridization.
Hybridization – Process where two complementary
single strands of nucleic acid (DNA or RNA) form a
double helix.
WB is a protein detection technique that combines
the separation power of SDS PAGE together with
high recognition specificity of antibodies
An antibody against the target protein could be
purified from serum of animals (mice, rabbits, goats)
immunized with this protein
Alternatively, if protein contains a commonly used
tag or epitope, an antibody against the tag/epitope
could be purchase from a commercial source (e.g.
anti-6 His antibody)
1. Separation of proteins using SDS PAGE
2. Transfer of the proteins onto e.g. a
nitrocellulose membrane (blotting)
3. Immune reactions
4. Visualization
This technique combines the technique IEF
(first dimension), which separates proteins
in a mixture according to charge (PI), with
the size separation technique of SDS-PAGE
second dimension).