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WHAT IS ELECTROPHORESIS?
A method of separating large molecules (such as DNA fragments or Proteins ) from a mixture of similar molecules.
An electric current-----passed through a medium containing the mixture----travels through the medium (Agarose and acrylamide gels) at a different rate-----depending----electrical charge and size.
DIFFERENT ELECTRPHORETIC TECHNIQUES:
Isoelectric focusing (IEF)
Western blotting / immunostaining
Pulse field gel electrophoresis
The pH------buffer conditions----arranged-----molecules being separated-----carry a net (negative) charge-----moved by the electric field toward the positive pole.
As they move-----the larger molecules----- will be held up-----smaller molecules----move faster.
This results in a separation by size----larger molecules------nearer the well-----smaller molecules ----farther away
Power = I2R I = current R= resistance
Mobility = applied voltage x net charge on the molecule / function of molecule (shape, size)
AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis-----preparation and analysis of DNA----gel slows the movement of DNA and separate by size.
DNA-----negatively charged----migrate toward the positive pole (anode)---mobility that is inversely proportional to the log10 of their molecular weight
Different concentrations of agarose----different sizes of DNA fragments.
Higher concentrations----separation of small DNAs
while low agarose concentrations----resolution of larger DNAs.
Estimation of the size of DNA molecules restriction mapping of cloned DNA.
Analysis of PCR products.
genetic diagnosis or genetic fingerprinting.
Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.
Easily cast and handled
nucleic acids are not chemically altered during electrophoresis.
Samples are also easily recovered.
The resulting gel can be stored in a plastic bag in a refrigerator.
Since passing current through a gel causes heating, gels may melt during electrophoresis.
Charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution.
Different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons
SDS (Sodium dodecyl sulfate) – PAGE (Poly Acrylamide Gel Electrophoresis)----to separate proteins----electrophoretic mobility (a function of length of polypeptide chain or molecular weight).
Proteins ----mixed with SDS-denatures secondary and non–disulfide–linked tertiary structures,-----negative charge ---in proportion to its mass.
A tracking dye may be added.
After electrophoresis----gel is treated with trichloroacetic acid solution----makes the proteins insoluble -----prevent them from diffusing during staining procedure
Gels are polymerized in a gel caster.
First the separating gel is poured and allowed to polymerize.
Next a thin layer of isopropanol is added.
Next the loading gel is poured and a comb is placed to create the wells.
After the loading gel is polymerized the comb can be removed and the gel is ready for electrophoresis.
Anode and cathode buffers are prepared.
The anode buffer----Tris-Cl, distilled deionized water ----higher pH—then cathode buffer.
The cathode buffer----SDS, Tris, Tricine, and distilled d
Negatively-charged proteins-----migrate towards the positive (+) electrode (anode).
Depending on their size----short proteins ----fit through the pores in the gel----larger ones---more resistance.
smaller proteins ---traveled farther down ----larger ones----remain closer to origin
After electrophoresis----gel is treated with ------trichloroacetic acid solution-----makes the proteins insoluble ----prevent them from diffusing during staining procedure
Comassie blue in methanol-acetic acid -----for staining
Polyacrylamide Gels are used to obtain high resolution separations for smaller molecules (STR analysis and DNA sequence analysis).
To evaluate genetic purity and diversity
Sds-page-- in the study of the epidemiology
Small pore size.
By changing the concentration of the monomer and the cross-linker, the pore size can be varied in a reproducible manner.
It is synthetic so no batch to batch differences.
Electrophoresis------ in a buffer filled, narrow-bore capillaries
Each capillary ------ 25-100 μm in internal diameter
When a voltage is applied ------the molecules move through the solution------ towards the electrode of opposite charge
Depending on the charge-----the molecules move through-----different speeds-----Separation is achieved
A photocathode -----used to measure the absorbencies of the molecule
The absorbencies----analyzed by a computer -----represented graphically
B Sivasankar (2007);Bioseparations: Principles And Techniques; Prentice-Hall Of India, New Delhi; Pp-240-254.
J. E. Corkill, P. R. Sisson , A. Smyth , J. Deveney, R. Freeman , P. Shears, D. Heaf and C. A. Hart (1993); Application Of Pyrolysis Mass Spectroscopy And SDS-PAGE In The Study Of The Epidemiology Of Pseudomonas Cepacia In Cystic Fibrosisj; Med Microbiol 41 ,pp-106-111.
Vo Cong Thanh, Pham Van Phuong, Phan Ho Hai Uyen, Phan Phuoc Hien(2006);Application Of Protein Electrophoresis Sds-page To Evaluate Genetic Purity And Diversity Of Several Varieties;Journal Of General Microbiology 80. pp-567-578.
A Geraldin,Willshaw, H. R. Smith And E. S. Anderson(1979) ;Application Of Agarose Gel Electrophoresis To The Characterization Of Plasmid DNA In Drug-resistant Enterobacteria; Journal Of General Microbiology (1979), 114,pp- 15-25.
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