ELECTROPHORESIS.pptx

electrophoresis

1. ELECTROPHORESIS

2. WHAT IS ELECTROPHORESIS? Electrophoresis is motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electro kinetic phenomenon was observed for the first time in 1807 by Reuss. There are several types of electrophoresis but the concept is same.

3. TYPES OF ELECTROPHORESIS HIGH RESOLUTION ELECTROPHORESIS AGAROSE GEL ELECTROPHORESIS POLYACRYLAMIDE GEL ELECTROPHORESIS ISOELECTRIC FOCCUSING ROUTINE ELECTROPHORESIS CAPILLARY ELECTROPHORESIS TWO DIMENSIONAL ELECTROPHORESIS IMMUNOCHEMICAL ELECTROPHORESIS IMMUNOFIXATION ELECTROPHORESIS ELECTROIMMUNOASSAY 9. PULSED FIELD ELECTROPHORESIS

4. GENERAL PRINCIPLE Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins , which can have either a net positive or net negative charge , nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.

5. ROUTINE ELECTROPHORESIS Routine electrophoresis is a generic term for the traditional clinical laboratory electrophoresis which is performed on a rectangle shaped slab of gel. It is mostly used for separation of protiens and has some use in separating nucleic acids. Generally several patient specimen and control can be placed on one gel and solute separated in one run. This electrophoresis is sometimes called as zone electrophoresis.

6. GEL ELECTROPHORESIS ELECTRO = flow of electricity PHORESIS = to carry across A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin. This refers to separation of charged particles located in a gel when an electric current is applied. Charged particles include DNA, RNA, amino acids, peptides etc.

7. Gel electrophoresis apparatus

8. STEPS 1.PREPARE AGAROSE GEL 2. ADD ETHIDIUM BROMIDE 3.POUR INTO CASTING TRAY 4. PUT GEL IN PLASTIC TRAY & ADD BUFFER 5. SAMPLES ARE PLACED IN GEL 6. START ELECTROPHORESIS 7.AFTER ELECTROPHORESIS IS COMPLETE READ UNDER UV LIGHT

9. FACTORS INFLUENCING ELECTROPHORESIS AGAROSE CONCENTRATION higher concentration of agarose facilitate separation of smaller DNA. VOLTAGE higher voltage cause larger DNA fragments to move larger distance.

10. 1 Plasmid Vector DNA 2 2 1 Add DNA Sample onto agarose gel lane 2 (DNA ladder is in lane 1) 3 NEGATIVE ELECTRIC CURRENT POSITIVE DNA bands Are separated by size 4 Dye added binds to DNA 5 DNA bands are exposed on film Under uv light DNA is visualized 1KB 500 bp 200 bp GEL ELECTROPHORESIS FOR DNA SEPARATION

11. POLYACRYLAMIDE GEL ELECTROPHORESIS SDS-PAGE ( sodium dodecyl sulfate polyacrylamide gel electrophoresis) widely used in biochemistry, molecular, forensic and genetics to separate proteins according to their electrophoretic mobility( a function of length/ molecular weight) separates protein only on basis of molecular weight and no other property.

12. PROCEDURE TISSUE PREPRATION

13. PREPARING ACRYLAMIDE GEL

14. POURING OF GEL

15. ELECTROPHORESIS

16. STAINING COOMASSIE BRILLIANT BLUE MOST COMMONLY USED DESTAINED WITH METHANOL/ACETIC ACID. 20-60 MINUTES SILVER STAINING IF THE STAIN IS LIGHT. DEVELOP WITH SODIUM SULPHATE/ FORMALDEHYDE

17. 1 PROTEIN 2 - - - - - - - - ADD SDS SDS binds to amino acid residue And gives uniform negative charge To protein. With heat protein is Linearized. 3 Add protein sample on SDS PAGE gel lane 2 1 2 4 NEGATIVE ELECTRIC CURRENT POSITIVE Protein bands Are separated by size PROTEIN ELECTROPHORESIS METHOD

18. ADVANTAGES Polyacrylamide forms gels with pores of a much more controlled and uniform size than does agarose. Consequently, polyacrylamide gels can be used to separate molecules that differ in size by as little as 2% of their molecular weight. The range of pore sizes possible is also much broader. Acrylamide is particularly superior when very small pore sizes are needed. It can be used to effectively separate small molecules (i.e. DNA oligonucleotides of 100 bases or less) Another advantage of acrylamide as a gel matrix is that it is much stronger than agarose , producing gels that do not tear as nearly as easily. It is also possible to load larger quantities of material onto acrylamide gels and to recover the material in a very pure form .

19. DISADVANTAGES Polyacrylamide gels are difficult to prepare when compared with agarose gels and the risk associated with their preparation is much greater. It is most dangerous in its powder form, when it is easily inhaled. Masks, gloves and safety glasses must always be worn when working with polyacrylamide powder.

20. USES SDS PAGE :- MOLECULAR BIOLOGY BIOCHEMISTRY GENETIC STUDY FORENSIC MICROBIOLOGY GEL ELECTROPHORESIS :- MASS SPECTROMETRY RFLP PCR CLONING DNA SEQUENCING SOUTHERN BLOT

21. ELECTROPHORESIS FOR SICKLE CELL ANAEMIA Pattern of hemoglobin electrophoresis from several different individuals. Lanes 1 and 5 are hemoglobin standards. Lane 2 is a normal adult. Lane 3 is a normal neonate. Lane 4 is a homozygous HbS individual. Lanes 6 and 8 are heterozygous sickle individuals. Lane 7 is a SC disease individual.

22. ELECTROPHORESIS FOR MULTIPLE MYELOMA The normal plasma protein electrophoretic pattern shows a heavy albumin band (to the left) and lighter bands which are alpha, beta and gamma mobility globulins. The serum from the patient with multiple myeloma shows a heavy band which is the abnormal monoclonal immunoglobulin (referred to as a paraprotein). Because it is monoclonal (all molecules identical) it is a very narrow discrete band, which in this patient has gamma mobility. α β γ

23. LIPOPROTEIN ELECTROPHORESIS CHYLOMICRON

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