PAPER ELECTROPHORESIS.pptx

Electrophoresis is a scientific laboratory technique that is used to separate DNA, RNA, or protein molecules based on their size and electrical charge. An electric current is passed through the molecules to move them so that they can be separated via a gel. The pores present in the gel work like a sieve, allowing smaller molecules to pass through more quickly and easily than the larger molecules. According to the way conditions are adjusted during electrophoresis, the molecules can be separated in the desired size range.

What is electrophoresis and what are its uses?
Electrophoresis is a very broadly used technique that, fundamentally, applies electric current to biological molecules – they’re usually DNA, but they can be protein or RNA, too – and separates these fragments into pieces that are larger or smaller in size.

The phenomenon of electrophoresis was first observed by Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuss in 1807 at Moscow University. A constant application of electric field caused the particles of clay dispersed in water to migrate, showing an electrokinetic phenomenon.

Electrophoresis can be defined as an electrokinetic process that separates charged particles in a fluid using an electrical field of charge. Electrophoresis of cations or positively charged ions is sometimes referred to as cataphoresis (or cataphoretic electrophoresis). In contrast, sometimes, the electrophoresis of anions or negatively charged ions is referred to as anaphoresis (or anaphoric electrophoresis).

It’s used in a variety of applications. Though it is most often used in life sciences to separate protein molecules or DNA, it can be achieved through several different techniques and methods depending upon the type and size of the molecules.

The methods differ in some ways, but all we need is a source for the electrical charge, a support medium and a buffer solution. Electrophoresis is also used in laboratories for the separation of molecules based on their size, density and purity.

The method used to separate macromolecules such as DNA, RNA, or protein molecules is known as gel electrophoresis.

It is used in forensics for –

Nucleic acid molecule sizing
DNA fragmentation for southern blotting
RNA fragmentation for northern blotting
Protein fragmentation for western blotting
Separation of PCR products analysis
Detection and analysis of variations or mutations in the sequence
Its clinical applications involve –

Serum protein electrophoresis
Lipoprotein analysis
Diagnosis of haemoglobinopathies and hemoglobin A1c.
The fundamental principle of electrophoresis is the existence of charge separation between the surface of a particle and the fluid immediately surrounding it. An applied electric field acts on the resulting charge density, causing the particle to migrate and the fluid around the particle to flow.

It is the process of separation or purification of protein molecules, DNA, or RNA that differ in charge, size.

1. M.Sc. – MICROBIOLOGY SEMESTER –II { INSTRUMENTATION S } PAPER ELECTROPHORESIS

2. CONTENT : 1] Introduction 2] Electrophoresis & its Principle 3] Types of electrophoresis [A] Free electrophoresis (1) Micro electrophoresis (2) Moving boundary [B] Zone electrophoresis (1) Paper electrophoresis (2) Cellulose electrophoresis (3) Gel electrophoresis 4]Paper electrophoresis (filter paper , apparatus, sample application, principle, electro phoretic run or working, detection & qualitative assay ) Its types on the basis of (1) Voltage applied – (a) Low voltage electrophoresis - Advantages & Disadvantages (b) High voltage electrophoresis - Advantages & Disadvantages (2) Design of instruments – (a) Horizontal paper electrophoresis (b) Vertical paper electrophoresis - Advantages & Disadvantages 5] Factors affecting paper electrophoresis 6]Application of paper electrophoresis 7] reference

3. INTRODUCTION : 1] The transport of particles through a solvent by application of an electric field is called as electrophoresis. 2] Most of the polymers (containing macromolecules) are electrically charged & will therefore move in an electric field. 3] Electrophoresis is useful in identification and structure determination of such big molecules. DEFINITION Electro means Electricity, Phoresis means Separation Separation of serum proteins ,amino acids , nucleic acid by the effect of an electric current. Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric change in conducting electrodes. Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an external electrical field. 4) Ions that are suspended between two electrodes tends to travel towards the electrodes that bears opposite charges

4. Macromolecules can be characterized by their rate of movement in an electric field. This property is used to determine protein molecular weights, to distinguish molecules by virtue of their net charge or their shape and to separate different molecular species quantitatively. Rate of movement of macromolecules in an electric field is useful parameter to know any change in amino acid regarding its charge. Electrophoresis is similar to chromatography. Electric field is used as a dragging force. Technique is simple, very effective and clean. Large number of samples can be separated, identified and quantitatively measured Principle MIGRATION OF AN ION IN AN ELECTRIC FIELD Consider a situation where a spherical molecule of net charge q is placed in an electric field. The force F which will act upon this particle will depend upon:- ( i ) the net charge density of the molecule (ii) the strength of the field in which it is placed.

5. F=∆E/d . q where ∆E/d is the field strength applied (∆E is the potential difference between the two electrodes, d being the distance between them). Since the particle has been suspended in a solution, we have to consider the friction occurring between the accelerating molecule and the solution, to arrive at a valid relationship of electrophoretic migration. The extent of the friction, as described by Stoke's equation, will depend upon ( i ) the size and shape of the molecule. (ii) on the viscosity of the medium through which the molecule will migrate. F=6 π r η v where F is the friction exerted on the spherical molecule, r is the radius of this molecule, η is the viscosity of the solution, and v is the velocity at which the molecule is migrating. This frictional force will oppose the accelerating force generated by the electric field . Equating the force of acceleration with Stoke's equation, we get

6. ∆E/d . q =6 π r η v If we rearrange the above relationship we get the expression V=∆ Eq /6 π r η d It will thus be seen that the velocity (v) of the molecule is proportional to ( i ) the field strength (∆Ed). (ii) charge (q) on the molecule . V is inversely proportional to :- the particle size (r). the viscosity of the solution ( η ).

7. Types of electrophoresis [ A] Free electrophoresis (1) Micro electrophoresis (2 ) Moving boundary electrophoresis [B] Zone electrophoresis (1 ) Paper electrophoresis ( 2) Cellulose electrophoresis ( 3) Gel electrophoresis zone electrophoresis In this method, an inert polymeric supporting media is used between the electrodes to separate and analyze the sample. The supporting media used in zone electrophoresis are absorbent paper, gel of starch, agar and poly acrylamide .

8. The major advantage of presence of supporting media is that it minimizes mixing of the sample and immobilization of the molecule after electrophoresis. It makes the analysis and purification of the molecule from the gel much easier than the moving boundary electrophoresis. The gel electrophoresis is the best example of zone electrophoresis. Methods of zone electrophoresis Paper electrophoresis Cellulose electrophoresis Gel electrophoresis Paper electrophoresis Paper electrophoresis (PE) is useful for the separation of small-charged molecules, such as amino acids and small proteins using a strip of paper (chromatography paper).

9. In this technique, the motion of colloidal particle of solution occurs leading to subsequent separation along the paper strip. PE is easier in comparison to gel electrophoresis. It does not require matrix preparation and it does not contain charges that interfere with the separation of compounds. Principle The charge carried by a molecules depend on the PH on the medium . Charged molecules are placed in an electric field, they migrate towards their respective positive & negative charge. CATHODE ( - ve ) = CATION ( + ve ) ANOD(+ ve ) = ANION ( - ve )

10. Factors affecting the paper electrophoresis The Sample- Charge- Higher the charge greater the mobilitySize - Bigger the molecule greater the frictional and electrostatic forces exerted on it by the medium . i.e. larger particles have smaller electrophoretic mobility compared to smaller particles. Electric field -I ncrease of migration with the increase of voltage gradient. Buffer- Migration of charge particle depend on of the buffer. a) composition - Commonly used buffers are Formate ", "Acetate", "Citrate", " Phosphate", " EDTA"The choice of buffer depends upon the type of sample being electrophoresed . b) PH : The extent of ionization depends on pH, especially in organic compounds. The ionization. increases with increase in PH of an organic acids and its just reverse for the organic bases therefore affecting its rate of migration. 4.TheMedium: The inert medium can exert adsorption ,molecular sieving effects & electro-osmosis - processes that affect the electrophoretic rate.

11. a) A dsorption: It means retention of a component on the surface of supporting medium. The rate and resolution of the electrophoretic separation can be efficiently reduced by adsorption. b) Molecular sieving: Media such as " Polyacrylamide ", "Agar", "Starch" & " Sephadex " have cross-linked structures giving rise to pores within the gel beads. 4 . Heat generation in electric fields - One of the practical problems encountered in electrophoresis is generation of heat from resistance in the electrophoretic medium. Heating not only changes viscosity and density of the electrophoretic media, it also damages equipment.

12. Instrumentation & apparatus [1] power pack [2] the electrophoretic cell [3] filter paper [4] sample (amino acid , proteins , nucleic acid ) [5] dyes Working or electrophoretic run A strip of filter paper is moistened with buffer and the ends of the strip are immersed into buffer reservoirs containing the electrodes. The samples are spotted in the center of the paper and high voltage is applied. • Application of high voltage causes less diffusion of small molecules giving better resolution and it take less time to complete the process. • Spots migrate according to their charges. • After electrophoresis, separated components can be detected by variety of staining techniques, depending upon their chemical composition

13. Types of paper electrophoresis On the basis of = [1] Voltage applied [A] Low voltage paper electrophoresis [B] High voltage paper electrophoresis [2]Design of instrument [A] Horizontal paper electrophoresis [B] Vertical paper electrophoresis Low & high voltage paper electrophoresis (battery ) [1] lvpe = 8-15 v/cm = 100-300 v (overall voltage across electrodes ) [2] hvpe = 50-215 v/cm = 10000 v ( overall voltage across electrodes ) Advantages of lvep Disadvantages if lvep 1. it is Simple & Safe technique 2. It is low cast – Technique 3. mare no. S a mple c an be Separated on a filter paper at a time

14. 1. Time Consuming techniqu e (mare the n 6-8 hrs) 2. Diffused Sample bands may appear. Advantages of hvep 1) Separation is fast. 2) Sh a rp bands are obtai n ed . 3) Mo re numbers of S a mple can be analyzed Similtaneously. Disadvantages of hvep 1)More V olt age is required to it is dangerous to operate. Horizontal paper electrophoresis

15. vertical paper electrophoresis

16. ADVANTAGES OF PAPER ELECTROPHORESIS 1. it is Sim ple , Easy & l ow cost technique. 2. Number of Samples can be applied on a paper at a time. Disadvantages of paper electrophoresis ☆ It is more time consuming technique 12-14 hus are required for Complete Separation of Samples.

17. Application of paper electrophoresis a simple, inexpensive, and accurate laboratory procedure for various research and clinical studies. easily available and easy to handle, allowing new methodologies to be tried and developed with convenience. Clinical applications of PE include study of sickle cell disease, hemoglobin ab normalities, and separation of blood clotting factors and serum plasmaproteins from blood sample . U sed in separation and identification of alkaloids. U sed for testing suitability of municipal water supplies, toxicity of water, and other environmental components. Drug-testing industry uses paper electrophoresis to determine presence of illegal or recreational drugs in job applicants and crime suspects. since 1950s used by the investigators and in forensics to analyze inks usedin currency to check the counterfeiters. Lack of sensitivity and reproducibility are limitations of PE.

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