## ELECTROPHORESIS

### ELECTROPHORESIS

18.      ELECTROPHORESIS

The word electrophoresis is derived from a Greek word, which means borne by electricity.

It is the basic technique for the separation of molecules in an electrical field. It is a separation method based on the differential rate of migration of charged species in an applied electric field. If molecules are supplied with an electric charge than all molecules are separated on the basis of its molecular weight. The rate of migration and separation depends upon :
(1)    Depends upon charge and size
The migration of biomolecules depends on their properties as well as the electrophoretic system
Based on Stoke’s law the mobility of particle (µ) can be calculated from
µ = Q/π r η
Where,
•    Q= charge of the particle
•    µ= mobility of the ion
•     r= radius of the particle in cm
•    η = viscosity of the medium
(2)    Separation based on the difference in charge to size ratio
Molecules which have higher molecular weight will migrate slowly due to higher frictional force of the molecule whereas molecules with low molecular weight migrate faster.
An ion with a charge (q) experiences a force (F) which is
F = Eq/d
where E = voltage
d = distance between the electrodes.

Agarose is most commonly used the material for gel formation (casting). Sometimes cellulose acetate and thin polyacrylamide gel are also used.  The sieve size of the gel is varied according to the concentration. Most commonly, electrophoresis is used for the separation of proteins and nucleic acids.
For electrophoresis several types of supports like paper, cellulose acetate, or gel composed of starch, agarose or polyacrylamide are used.
18.1.    The Rate of migration of particle depends on-
(1)    The strength of the field – For migration of molecules electric voltage supply is the must. As the voltage supply is increased it will increase the movement of sample molecules and fast movement will be observed.
The migration of particle in the electric field is directly proportional to the field intensity (in V/cm).
Note: If the supply is increased above an appropriate limit than it can melt the gel due to the production of heat.
(2)    The shape of the molecules – Electrophoretic mobility is greatly related to the shape and size of molecules. As higher the molecular weight of molecules they will move slower and lower molecular weight causes faster movement of molecules.
The shape of molecules affects the migration as linear molecules (Large size) will move slower whereas compact globular molecules (small size) moves faster.
Pore size – Pore size of the gel can be controlled according to the requirement of the experiment. As higher will be the concentration of gel, it will form compact structure and pore size will be small and the mobility is reduced.

Whereas lower concentration of gel will make large pores and easier movement of molecules.
DNA and RNA always move from cathode towards anode because the presence of phosphate groups in the structure of DNA and RNA provides a negative charge to these molecules whereas proteins are ampholytes, i.e. they bear both positive and negative charge groups and the pH of the medium will determine the resulting net charge on the protein. So every protein has a specific isoelectric point at which it becomes neutral.
1.    Gel electrophoresis can be used for separation of larger samples than paper electrophoresis system.
2.    The character of the gel matrix can be altered according to the particular application.
Types of Gel electrophoresis:-
18.1.1.    Agarose gel electrophoresis
It is a type of Horizontal direction electrophoresis. Agarose is the linear carbohydrate polymer which is extracted from seaweed, arabinose it forms porous matrix as it solidifies to form gel Agarose is a purified form of agar. It is a polysaccharide with repeating 1, 3 $\beta$ D galactopyranose and 1, 4-3, 6 unhydro L-galactopyranose residues obtained from agar. It is mostly used for separation of nuclucacls RNA and DNA at low concentration (0.8% to 3%). Large molecular weight proteins can also be separated on the basis of net charge difference only.

DNA and RNA both are negatively charged so during electrophoresis they gel separated by migrating separates from negative to positive terminal electrode.
For the preparation of gel, agarose is dissolved in a buffer solution and boiled till a clear transparent solution is obtained. After boiling the solution it will transfer in gel casting tray and place comb for wells formation. At 55°C, it starts to polymerization and converts in gel form. The gel form contains pores for the moment of molecules.
The pore size depends on the concentration of agarose.

Concentrations of agarose used
% Agarose (w/v)Size Range (kb pairs) for Optimal Separation
0.5        2 - 30 kb
0.75      0.7 - 20 kb
1.0        0.5 - 10 kb
1.5        0.2 - 3 kb
2.0        0.1 - 2 kb
Buffer solution and other chemicals
EtBr :
Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA under the UV light. It absorbed the UV light and emits fluorescent light (orange color).
After the separation of DNA, it visualizes in form of the band. See the DNA bands we have to use stain. Ethidium bromide is a chemical used for visualizing DNA bands in the gel. EtBr is an intercalating agent between stands of DNA. It is mix with warm liquid.

Buffer
TAE buffer and TBE buffer are used for Agarose gel electrophoresis. This buffer solution basically subsists the changes in the pH of gel and medium.
TAE - Tris base, Acetic acid, EDTA
TBE - Tris base, Boric acid, EDTA
Tracking dye :
For check the proses of gel electrophoresis separation. We use tracking dyes these dyes mixed with Baoding buffer. It migrates at a specific speed in different gel concentration. They did not intricate with DNA and not affected the separation of DNA generally they run ahead from the fragment the proses of electrophoresis is monitoring by movement of tracking dye. Usually, the dye is used for tracking are Xylene cyanol, Bromophenol Blue.
18.2.    PAGE (PolyAcrylamide Gel Electrophoresis)
It is the most common method of analysing the purity of an isolated protein. It is used to separate and characterise protein. Proteins are denatured by treatment with a strong anionic detergent sodium dodecyl sulfate (SDS). It is a amphipathic detergent. In this method, the free radical initiator, ammonium persulfate (APS), is added along with tetramethylethylenediamine (TEMED) catalyst. TEMED is a free radicle stabilizer which promotes polymerisation. In the presence of SDS, the parameters of shape and charge become unimportant and separation is achieved only on the basis of protein molecular weight.

18.2.1.    Chemical ingredients
18.2.1.1. Tris HCL (tris hydrochloric acid) used as a buffer. It is the solution containing tris-base with HCL.
18.2.1.2. Glycine (Amino Acetic Acid) it is a small molecule with no net charge on its surface so that it is positively charged when placed in acidic gel and negatively charged in the basic gel so that it is useful for the better resolution of protein bands.
18.2.1.3. Acrylamide is a white odourless crystalline powder. When this powder is dissolved in water it starts to autopolymerize and forms a gel. Normally these molecules are polymerized in linear form and due to the presence of free radicals they are activated as free ions and polymerize quickly as linear chains, the process known as vinyl addition polymerisation. (Free radicle polymerization).
18.2.1.4. Bisacrylamide (N, N1-Methylenebisacrylamide) it is used as a cross-linking agent for polyacrylamide gels which cross linked them and forms a stable gel.

18.2.1.5. Sodium Dodecyl Sulfate (SDS)  is used as a denaturing agent for native proteins. This detergent wraps around the polypeptide backbone and binds with it so providing a uniform negative charge to polypeptide chain masking all the charges of protein and breaks all the disulfide bonds present in protein molecules.

We can use BME (b-mercaptoethanol) to break disulphide bonds. In the absence of SDS, proteins will move on the basis of mass to charge ratio and cannot give accuracy in results because that protein can be multiple subunits, which is a drawback of native PAGE but after SDS treatment they will move only on the basis of molecular weight which solves this problem.

18.2.1.6. Ammonium persulfate (APS) It provides free radicals for the reaction and activates TEMED. Which in turn helps in the polymerisation of gel.
18.2.1.7.  TEMED (N, N, N', N'-tetramethylethylenediamine) acts as a catalyst for the polymerization of gel.
APS and TEMED are the main components for the polymerization for gel. The higher amount of APS and TEMED is in increases turbidity of gel and the length of polymers is decreased. Normally 1 to 10 mM concentration of  APS and TEMED are used.
The pore size of the gel is determined by two factors
1.    The total amount of acrylamide present (designated as %T)
2.    The amount of cross-linker (%C). (bis-acrylamide)
Mostly 5% of the crosslinker is used as for smallest pore size and as the concentration of crosslinker is increased it starts to incorporate between the acrylamide linear chains causing the increase in the pore size of the gel.
18.3.    Chemicals for visualization
•    Bromophenol blue (BPB) (3',3",5',5" tetra bromo phenolsulfonphthalein) is the most popular gel tracking dye. It gives a blue colour to the samples. Proteins and nucleic acids are colourless so when they are electrophoresed then it is necessary to watch their distance migrated otherwise they will cross the gel and get destroyed in the buffer.
•    Coomassie Brilliant Blue is mostly used for staining of protein samples. It is non-polar, an anionic dye, which binds with proteins non-specifically.
Types of Gel -
Two types of gels are used which is partitioned into two sections, known as, the stacking Gel and the resolving  Gel (running Gel). Electricity is passed through the gel by the electrodes, so generally at the stacking gel and cathode is present and towards the resolving gel it is the anode. When electricity is passed through it samples starts to move from cathode to anode because of the negative charge on their surface.

18.4.    Application
-    Used to identify any specific protein in the given sample.
-    Quantify the proteins.
-    Analysis of protein spectra of body fluids.
-    Identification of proteinuria i.e. protein present in urine in abnormal quantities.
-    Identification of subunits of proteins

18.5.    Isoelectric Focusing
This technique separates proteins molecules which are focused on a point where pH=pI in an applied electric field. This technique separates molecules according to their charge. Every protein molecule has a specific charge on its surface and this charge is neutralized at a specific pH. So that point where proteins have a neutral charge is termed as isoelectric point (pI).  At this point, proteins don’t move further. Therefore PI is that pH at which charge on protein will be zero.

Figure:- showing that at low pH the NH2 group accepts H+ ion and converts to NH3+ and provide positive charge on protein whereas at high pH the COOH group loses its H+ group and gives a negative charge to protein and at pI protein have both charges which neutralize protein.
PI > pH    =    Positive charge
PI < pH    =    Negative charge
For separation of proteins solution of ampholytes is used molecules which contains both positive and negative charge is passed through the column so a pH gradient is formed throughout the gel. The sample of proteins is electrophoresed through gel and migration of molecules is seen in the gel according to its charge. Molecules can be resolved up to 0.02 pH difference.
2 – D Gel Electrophoresis (Two-dimensional Gel Electrophoresis) :

This technique named so because of movement of proteins in two dimensions as one dimension in isoelectric focusing which separates protein according to their PI which runs horizontally and in SDS-PAGE which separates according to molecular weight and runs vertically. Proteins get resolved on the basis of charge in isoelectric focusing according to pI in a tube. The gel is then removed and put on the top of an SDS-PAGE slab. Now proteins are resolved according to their molecular weight.
18.5.1.    Applications :
Characterization of proteins to be separate and resolves proteins from a protein sample. This technique is used for many pharmacological studies.
Characterizing Protein Modifications- Post-translational modification can make the change like phosphorylation, glycosylation, acylation in proteins and alter the molecular weight, pI of proteins.
Characterization of mutant proteins----Identification of mutant proteins in the cell system and study of the effect of that protein with the surrounding proteins.
-    Identification of proteins involved in the toxic response
-    Involves metabolic pathway manipulation
-    Study of protein-protein interactions
18.6.    Pulse Field Gel Electrophoresis (PFGE)
This technique is used to fractionates large DNA molecules ranging from 10 kb to 10 Mb by electrophoresis in agarose gel in two directions. This Concept was first described by Schwartz (1982). It resolves DNA by alternating the electric field between the pair of electrode.

The principle of PFGE is that large DNA fragments require more time to reverse the direction in an electric field compared to small fragments.
The equipment used in this technique is comprised of 6 – 8 set of electrodes in the gel stack. Then the direction of the electrical field is repeatedly changed in the gel. The sample of the genome is the first restriction digested which generates short fragments of nucleotides. Now, these fragments are electrophoresed in the equipment.
18.6.1.    Types of PFGE
(1)    Field-Inversion Gel Electrophoresis (FIGE) – In this technique electrode polarity is reversed at intervals so a net forward migration is achieved by the difference in change in the reversal of polarity. It is also named as switch time ramping.
(2)    Transverse-Alternating Field Gel Electrophoresis (TAFE) – The gel is oriented vertically and a four-electrode combination is used in the gel so that molecules move in a zig-zag fashion.
(3)    Contour-clamped Homogeneous Electric Fields (CHEF) – This apparatus provides a more complex solution for separation of molecules.
(4)    Rotating Gel Electrophoresis (RGE) - in this method gel is rotated between two set angles and a uniform electric field is applied so that effect by different angles is also studied.
(5)    Programmable Autonomously-Controlled Electrodes (PACE) – This method controls overall electric field and regulation over 24 electrodes.

18.6.2.    Application:-
Used for genotyping or genetic fingerprinting. Genome characterization and construction of the physical map. YAC libraries formation. Easy isolation of the individual restriction fragments for restriction mapping, gene insertion and functional gene mapping. Genome size estimation and the construction of chromosomal maps. To study DNA damage and repair, size organization and centromere variation. To study the degree of relatedness among different strains of the same species. It allows manipulations with DNA of whole chromosomes or their large fragments. In identifying the genetic defects that cause many hereditary diseases.
18.7.    Denaturing Gradient Gel Electrophoresis (DGGE)
These are the special type of gel electrophoresis in which a gradient of the physical factor is applied to the molecule which affects the integrity of that molecule. Temperature gradient gel electrophoresis temperature gradient is formed whereas in denaturing gradient gel electrophoresis denaturing agent gradient is formed. Biological molecules like DNA, proteins and RNA have the different capability of movement according to their charge, shape and molecular weight. So they are resolved by gel electrophoresis.
In DGGE, a gradient of the denaturing agent of molecules like urea, formamide is created in the gel. So as the molecule is gel electrophoresed it starts to move in the gradient and when it reaches the threshold concentration of denaturing reagents it starts unbinding and said to be melted. Likewise In TGGE temperature gradient is formed which also melts the DNA after a threshold limit. Basically, the reason behind the melting of these molecules is that hydrogen bonding between the base pairs is broken by the temperature and denaturing agents. So as the concentration is increased molecules gets distorted and the migration of molecule is decreased. In nucleotide, a higher G:C content is more stable than the higher A:T content because of its 3 hydrogen bonds between nucleotide.
Three Steps in DGGE/ TGGE

18.7.1.    Applications:-
Determination of relationship among microorganisms and their environment. Detection of numerous mutations like in mitochondrial DNA, p53 genes etc. Study of complex microbial communities such as the gastrointestinal (GI) tract of food-producing animals. Diagnosis of emerging infections which are not able to detect in other gel electrophoresis techniques. Distinguish between mutated and wild type sequences.