Agarose and Polyacrylamine Gel Electrophoresis Khrystall K. Ramos CallejasDepartment of Biology Gretel S. Montañez PróspereUniversity of Puerto Rico at Cayey Luis Pérez SotoRISE Program Wilmarie Morales SotoMarch 16, 2012
WHAT IS GEL ELECTROPHORESISElectrophoresis is the term used for the procedure where under the influence of voltage, a charged particle moves.It is a standard method for separation, identification, analysis and purification of: DNA molecules protein molecules
Electrophoresis consists of the migration of a charged molecules under the influence of electric field (from negative to positive).A buffer solution is use to conduct electricity through the whole setup of the gel electrophoresis.The molecule will migrate through the gel depending upon the size and shape.
Gel electrophoresis is used: Forensics Molecular biology Genetics Microbiology BiochemistryThe results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device; analyzing the intensity of the band or the measure of the spot of interest.
TYPES OF GELS:1. Agarose*2. Polyacrylamide*3. Starch
AGAROSE GEL ELECTROPHORESISAgarose is a linear polymer extracted from seaweed that forms a gel matrix by hydrogen- bonding when heated in a buffer and allowed to cool.The agarose gel is used to separate DNA and RNA fragments.Agarose gels separate DNA fragments differing by a hundred or more base pairs.
DNA has negative charge so it migrates towards the positive end.This is due to its double helical physical structure, which contains a phosphate backbone.
The density and porosity of the gel matrix is determined by the concentration of agarose used.The grater the agarose concentration, the smaller the pores created in the gel matrix, the more difficult it is for larger DNA molecules to move through. Agarose % Optimum Resolution for DNA 0.5 1,000-30,000bp 0.7 800-12,000bp 1.0 500-10,000bp 1.2 400-7,000bp 1.5 200-3,000bp 2.0 50-2,000bp
POLYACRYLAMIDE GEL ELECTROPHORESISLike Agarose Gels, Polyacrylamide gels are used to separate protein molecules by shape, size and charge.Polyacrylamide is a polymer of acrylamide monomers.
Polyacrylamide is specifically used for proteins because it provides the protein with an environment where it will not become denatured.Allowing different sized proteins to move at different rates.
Since we are trying to separate many different protein molecules of a variety of shapes and sizes, we first want to get them to be linear so that the proteins no longer have any secondary, tertiary or quaternary structure.
To have proteins with linear structures we use sodium dodecyl sulfate (SDS).SDS is a detergent that can dissolve hydrophobic molecules, resulting in proteins with linear structures.
Another problem we face with proteins is that they do not have a specific charge.This is another reason why SDS is important. SDS has a negative charge and by dissolving the protein in it, the protein becomes negatively charged.Allowing it to run properly through the gel (from negative to positive).
Get your sample obtained from previous purifying technique (i.e. PCR) Load BufferSet up gel, removecomb Load Sample Run GelStain and look at withUV light
APPLICATIONS OF GELS:Estimation of the size of DNA and protein molecules.Analysis of PCR products, i.e. in molecular genetic diagnosis or genetic fingerprintingSeparation of restricted genomic DNA or of RNA.