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Electrophoresis Editted
Electrophoresis Editted
lecture Outline
1- Introduction.
2- Principle.
3- Factors affecting the rate of migration . 4- solutions. 5- Principal of electro-endosmosis. 6- Factors affecting electrophoresis mobility. 7- Instrumentation. 8-Types of electrophoresis.
Introduction
The term electrophoresis describes the migration of a
,peptides ,proteins ,nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations (+) or anoins (-).
Under the influence of an electric field these charged
particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.
Principle
Any charged ion or molecule migrates when placed in an electric field. The rate of migration depend upon its net charge, size, shape , pH , properties of the support medium, time frame of the procedure, temp. of the operating system and the applied electric current. This can be represented by following eq.: E*q V = --------------f
v = velocity of migration of the molecule. E = electric field in volts per cm q = net electric charge on the molecule f = frictional coefficient
The movement of charged particle in an electric field is expressed in terms of electrophoretic mobility ,denoted by . where,
= q/f
For molecules with similar shapes, f doesnt vary however, it varies with size. Thus electrophoretic mobility () of a molecule is directly proportional to charge density(charge\mass ratio).
sample reach the electrodes, the components will have been separated according to their electrophoretic mobility. Thus, electrophoresis is an incomplete form of electrolysis . The sample then are then located by staining with an appropriate dye.
The current in the solution between the electrodes is
conducted mainly by the buffer ions, a small proportion are being conducted by the sample ions. Ohms law express the relationship between current (I),voltage (V) and resistance ( R ): R = V/I.
electrophoretic separation by increasing the applied voltage, which would result in a corresponding increase the electric flowing . The distance migrated by the ions will be proportional to both current and time.
charge of a molecule/atom.
5- prosperities of support medium: A higher pore size will increase mobility while, increase
to heat. This may include denaturation of proteins (and thus the lose of enzyme activity).
D- A decrease of buffer viscosity, and hence a reduction
solutions
1- Using a stabilised power supply which provides
final factor affecting separation is called electroendosmosis which is due to presence of charged groups on the surface of support medium .
Principal of electro-endosmosis
The static support, the stabilizing medium (e.g. the gel)
and/or the surface of the separation equipment such as glass plates, tubes or capillaries can carry charged groups: e.g. carboxylic groups in starch and agarose, sulfonic groups in agarose, silicium oxide on glass surfaces. These groups become ionized in basic and neutral buffers: in the electric field they will be attracted by the anode. As they are fixed in the matrix, they can not migrate. This results in a compensation by the counter flow of H3O+ ions towards the cathode: electroendosmosis.
cathode, which carries the solubilized substances along. The electrophoretic and electroosmotic migrations are then additive. The results are: blurred zones, and drying of the gel in the anodal area of flatbed gels.
When
fixed groups are positively charged, electroosmotic flow is directed towards the anode.
the
Instrumentation
1- Power back which supply DC. 2- Electrophoresis unit (for running either horizontal or
vertical ) : two glass plates (different pore sizes), plastic spacers, buffer and comb.
FREE ELECTROPHORESIS
ZONE ELECTROPHORESIS
MICRO ELECTROPHORESIS
MOVING BOUNDARY
PAPER ELECTROPHORESIS
It is the form of electrophoresis that is carried out on filter paper. This technique is useful for separation of small charged molecules such as amino acids and small proteins. FILTER PAPER : It is the stabilizing medium. We can use Whatman filter paper, cellulose acetate filter paper or chromatography paper.
applications. It has an advantage over paper in that it is a much more homogeneous medium, with uniform pore size, and does not absorb proteins in the way that paper does. It gives better resolution than paper electrophoresis ,however nothing as good as that achieved with polyacrylamide gels. It is especially useful for separating alpha immunoglobulins from albumin.
GEL ELECTROPHORESIS
It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic acid or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix.
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: 1- Starch gel 2- Agarose gel. 3- Polyacrylamide gel.
Starch gel : An early form of gel . Agarose gel :A highly purified uncharged polysaccharide
derived from agar. Used to separate macromolecules such as nucleic acids, large proteins and protein complexes. Percentage used (1-3%). Polyacrylamide gel (PAGE) : Used to separate most proteins and small oligonucleotides because of the presence of small pores. Percentage used (3-30%).
Example:
PCR products of the C-terminus of killifish CFTR gene before and after PCR purification. Both products were DNA electrophoresed in 3% Agarose gel. Bands in the first lane and third lane are referring to PCR product before and after PCR purification respectively. An estimation of PCR products sizes was determined by using DNA 100 bp marker in the second lane.
containing SDS and -mercaptoethanol. Protein gets denatured and opens up into rod-shaped structure. Sample buffer contains bromophenol blue which is used as a tracking dye, and sucrose or glycerol. Before the sample is loaded into the main separating gel a stacking gel is poured on top of the separating gel.
Cont.
Current is switched on.
continue to move towards the anode. As they pass through the separating gel, the proteins separate, owing to the molecular sieving properties of the gel. When the dye reaches the bottom of the gel, the current is turned off. Gel is removed from between the glass plates and shaken in an appropriate stain solution. Blue colored bands are observed under UV rays.
Example
The Purification check of the C-terminus of Killifish CFTR. Purification was checked using 15% SDS-PAGE gel. The Prestained Protein Marker in the first lane was used for molecular weight determinations. The lanes from (2-5) refer to IMAC unbound materials; washes and elutions before thrombin cleavage while lanes from (6-9) refer to IMAC unbound materials; washes and elutions after thrombin cleavage. The arrow in lane 6 refers to the purified c-terminus peptide.
TWO-DIMENSIONAL ELECTROPHORESIS
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).
dimension(2-D)PAGE provides a highly sophisticated analytical method for analysing protein mixtures.
paper to produce a sandwich of gel and nitrocellulose that compressed in a cassette and immersed in a buffer between parallel electrodes. when the current is passed it cause separating of protein from gel to nitrocellulose sheet which will examine further by using antibodies (specific to tested protein) , incubation ,second antibodies with marker and then expose blot to UV light.
2. Glycoproteins
Sudan black
4. Enzymes Appropriate enzymatic methods 5. Nucleic acids Ethidium Bromide or SYBR gold
Capillary electrophoresis
Used to separate wide range of biological moleculed
including : A.A , peptides, proteins, DNA fragments , metal ions and drugs. It involves electrophoresis of sample in a very narrowbore tubes. It is a highly speed analysis (20 mins ). It is a type of zone electrophoresis.
Microchip electrophoresis
Very high speed analysis (tens of seconds).
Use laser induced fluorescence. It required very low volts.
Clinical applications
1- Specific protein electrophoresis.
classes. 3- Separation and quantification of lipoprotein and lipid classes. 4- Separation and quantification of enzymes to its subtypes. 5- Separation and quantification of immunoglobulins. 6- Use of western blot tech./ southern blot tech.