Electro phoresis

what is electrophoresis
polyacrylamide gel electrophoresis

1. ELECTROPHORESIS Presented by :- Patel Vanita L . CBO :- 406 M.Sc Sem :- 2 Department of life sciences , H.N.G.U., Patan

2. CONTENT What is electrophoresis ? Polyacrylamide gel electrophoresis 1. Native page 2. SDS Page

3. What is electrophoresis ? Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size . The technique applies a negative charge so proteins move towards a positive charge . This is used for both DNA and RNA analysis . Electrophoresis is an analytical method frequently used in molecular biology and medicine . It is applied for the separation and characterization of proteins, nucleic acids, and sub cellular sized particles like viruses and small organelles .

4. It’s Principle is that charged particles of sample migrate in an applied electric field. Type of electrophoresis :- Polyacrylamide gel electrophoresis 2- D Gel electrophoresis Paper electrophoresis Immuno electrophoresis

5. Polyacrylamide Gel electrophoresis :- Electrophoresis techniques separate charged molecules in an electric field . The mobility of a molecule in inversely proportional to its size and directly proportional to its charge . During Electrophoresis proteins move towards an oppositely charged electrode in an electric field . Polyacrylamide gel electrophoresis is a technique based on this idea and is used to separate proteins on the basis of their size .

6. Two type of PAGE : Native PAGE 2. SDS PAGE 1 . Native PAGE :- Principle :- In Native PAGE protein mixture are separated on T he bases of their molecular weight under the influence of applied voltage this are is principle of Native PAGE .

7. Native( Buffers ) Gel :- In Native or buffer gels polyacrylamide gels are again used ( normally a 7.5% gel ) but the SDS is absent and the proteins are not denatured prior to loading . Since all the proteins in the sample being analysed carry their native charge at the pH of gel (normally oh 8.7 ) protein separate according their different electrophoretic mobilities and the sieving effects of the gel . It is therefore not possible to predict the behaviour of a given protein in a buffer gels, but because of the range of different changes and size of protein in a given protein mixture , good revolution in achieved .

8. An alternative method for enzyme detection is to include the substrate in an agarose gel that is poured over acrylamide gel and allowed to set . Diffusion and interaction of enzyme and substrate between the two gels results in colour formation at the site of enzyme . Often , duplicate sample will be run on gel , the cut in half and one half stained for activity the other for total proteins . In this way the total protein content of the sample can be analysed and the particular band corresponding to the enzyme identified by reference to the activity stain gel .

9. SDS (Sodium dodecyl sulphate ) PAGE :- SDS PAGE is the most widely used method for analysing protein mixtures qualitatively . It is Particulary useful for monitoring protein purification and because the method is based on the separation of protein according to size it can also be used to determine the relative molecular mass of proteins .SDS is anionic detergent . Each protein in the mixture is therefore fully denatured by this treatment and opens up into rod-shaped structure with a series of negatively charged SDS molecules along the polypeptide chain . The original native charge on the molecule is therefor completely swamped by the negatively charged SDS molecules .

10. Once the samples are all loaded a current is passed thorough the gel . The sample to be separated are not in fact loaded directly into the main separating gel . When the main separating gel has been poured between the glass plate and allowed to set shorter stacking gel is poured on top of the separating gel and it is into this gel that the wells are formed and proteins loaded . The purpose of this stacking gel is to concentrate the protein sample into a sharp band before it enters the main separating gel .

11. This is achieved by utilising differences in ionic strength and pH between the electrophoresis buffer and involves a phenomenon known the istachophoresis . The stacking gel has a very large pore size which allows the proteins to move freely and concentrate or stack under the effect of the electric field . Small proteins migrate faster through the gel under the influence of the applied electric field .

12. Application for PAGE :- Separation of molecules . Prediction of molecule weight of separated molecule . You can study separate isoenzyme by use PAGE . This Technique is utilize of separation of the protein .

13. REFREENCES Principle and techniques of biochemistry and Molecular biology : by :- Keith Wilson John Walker https://www.ncbi.nlm.nih.gov

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