ELECTROFORESIS.

dr. ajit surya singh

1. Electrophoresis Dr. Ajit Kumar Singh PGT 1 st year (Laboratory medicine) Chittaranjan National Cancer Institute , Kolkata Moderator Dr. Garima Chauhan MD Biochemistry Assistant professor Department of Laboratory medicine Chittaranjan National Cancer Institute , Kolkata

2. General principles of electrophoresis Electrophoresis is the migration of charged particles or molecules in a medium under the influence of an applied electric field. A separation technique Simple, rapid , and highly sensitive Used in clinical laboratories to separate charged molecules : Proteins in body fluids : serum ,urine , CSF Proteins in erythrocytes : Hemoglobin Nucleic acid : DNA , RNA

3. General principles of electrophoresis

4. General principles of electrophoresis

5. General principles of electrophoresis Force experienced by particle in an electrical field is given by Coulomb’s law : F = ZqE ………………………………………… equation (1) The viscous resistance of the medium to the motion given by Stokes’ law : F’ = 6 π r η v …………………………………….. Equation (2) The viscous resistance of the medium just balances the driving force : F = F’ ZqE = 6π r η v Electrophoretic mobility (U) = v/E = Zq / 6π r η

6. Factors affecting electrophoresis Inherent factors Net charge of the molecules Size and shape of the molecules Molecular weight External environment Solution pH Electric field Solution viscosity

7. Instrumentation Power supply and chamber Buffer Support media Stain and Quantification (densitometer )

8. 1. Power supply and chamber

9. 2. Buffer The buffer in electrophoresis has two fold purpose Carry applied electrical current They set the pH at which electrophoresis is carried Tris acetate EDTA (TAE) and tris borate EDTA (TBE) (Most common used buffer) Buffer pH value Phosphate buffer around 7.0 Tris -Glycine buffer (TG) more than 8.5 Tris -Citrate-EDTA buffer (TCE) around 7.0 Tris -EDTA buffer (TE) around 8.0 Tris -Maleic acid -EDTA buffer (TME) around 7.5 Lithium Borate - buffer (LB) around 8.6

10. 2. Buffer The buffer in electrophoresis has two fold purpose : Carry applied electrical current They set the pH at which electrophoresis is carried out. Thus they determine : Type of charge on solute. Extent of ionization of solute Electrode towards which the solute will migrate. The buffer ionic strength will determine the thickness of the ionic cloud.

11. 3. Support Media The support medium provides the matrix in which protein separation takes place. Various types of support media have been used in electrophoresis and range from pure buffer solutions in a capillary to insoluble gels or membranes of cellulose acetate. - Starch gel - Cellulose acetate - Agarose - Polyacrylamide gel (PAG)

12. 4. Stain Separation type Stain Serum proteins Amido Black (Naphthol Blue Black) Coomassie Brilliant Blue (brilliant blue G) Coomassie Brilliant Blue (brilliant blue R) Ponceau S Isoenzymes Nitrotetrazolium Blue Lipoprotein Fat Red 7B (Sudan Red 7B) Oil Red O Sudan Black B DNA fragments Ethidium bromide CSF proteins Silver nitrate Depends on nature of the molecule to be detected

13. 5. Quantification A densitometer is used . Essential features of a densitometer include : The ability to scan gels 25 to 100 mm in length Variable wavelength control over the range of 400 to 700 nm An integrating device with both automatic and manual selection Automatic indexing

14. Conventional electrophoresis

15. DNA is negatively charged. When placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size.

16. Horizontal gel electrophoresis Agarose gel electrophoresis is a method to separate DNA , or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field ( electrophoresis ). Shorter molecules move faster and migrate farther than longer ones .

17. At any given P H , exist in a solution as electrically charged species either as a cation (+) or anion(-). Under the influence of an electric field these charged particles will migrate either to cathode or anode, depending on the nature of their net charged Electrophoresis is the movement of molecules by an electric current. Nucleic acid moves from a negative to a positive pole.

18. Vertical gel electrophoresis

19. Steps followed in gel electrophoresis Mix agarose and buffer Boil mixture in microwave Cool the mixture at 65 and pour in to chamber , comb inserted in to chamber to make well Gel solidified at room temperature , comb remove wells remain Load the prepared samples in to gel Separate fragments by electrophoresis Stain separated molecules and measure distance

20. Electrophoretic Separation When electrophoresis is performed following steps are typical Excess buffer is removed from the support surface by blotting , taking care that bubbles are not present 5 to 7 µL of sample is applied using a comb or a plastic template and is allowed to diffuse into the gel; it is then blotted to remove the excess The gel is placed into the electrode chamber Electrophoresis is performed at specified current, voltage, or power The gel is fixed, rinsed, and then dried The gel is stained and redried and The gel is scanned in a densitometer

21. Types of electrophoresis 1.Zone electrophoresis Discrete zone formed Example – Paper electrophoresis Agarose gel electrophoresis Poly acrylamide gel electrophoresis (PAGE)

22. Types of electrophoresis 2.Moving Boundary Electrophoresis Involves the migration of charged molecules in a free moving solution, without the presence of a supporting medium Example – Capillary electrophoresis Microchip electrophoresis

23. Types of electrophoresis 1. Zone Electrophoresis Paper electrophoresis Agarose gel electrophoresis Poly Acrylamide Gel Electrophoresis (PAGE) Pulsed-field gel electrophoresis (PFGE) SDS-PAGE 2D electrophoresis Immuno-electrophoresis(Rocket Electrophoresis) I . Difference Gel Electrophoresis(DIGE) 2.Moving Boundary Electrophoresis a) Capillary Electrophoresis b) Iso- tachophoresis

24. A. Paper electrophoresis Used in clinical investigations of serum and other body fluid Disadvantage : Adsorbs proteins Poor conductivity Background staining OH groups of cellulose bind with proteins and retard electrophoretic movements causing trailing of bands and poor resolution Advantage : It is economical , Easy to use , non-toxic , can be stored easily

25. A. Paper electrophoresis

26. Agarose gel A linear polysaccharide (made-up of repeat unit of agarobiose -alternating unit of galactose and 3,6-anhydrogalactose). Used in conc as 1% and 3%. The gelling property are attributed to both inter- and intramolecular hydrogen bonding. Pore size is controlled by the % of agarose used. Purity of the agarose is based on the number of sulphate conc, lower the conc of sulphate higher is the purity of agarose. Acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments

27. Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer. Agarose

28. Melting the Agarose ). Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right).

29. B. Agarose gel electrophoresis Agarose gel - porous material derived from red seaweed . Gel material acts as a "molecular sieve” and electrically neutral . Concentration affects DNA migration , used in conc as 1% and 3%. Low conc. = larger pores better resolution of larger DNA fragments High conc. = smaller pores better resolution of smaller DNA fragments

30. B. Agarose gel electrophoresis Effective separation range of agarose gels of various compositions for separation of nucleic acids - % Agarose ( wt / vol) Effective Separation Range (base pairs) 0.8 700–9000 1.0 500–7000 1.2 400–5000

31. C. Poly acrylamide gel electrophoresis (PAGE) Used up to 3-30% concentration lower concentration for DNA separation and higher concentration for protein separation

32. C. Poly Acrylamide Gel Electrophoresis (PAGE) % Acrylamide in Resolving Gel Effective Separation Range (Da) 7.5 45,000–200,000 10 20,000–200,000 12 14,000–70,000 15 5,000–70,000 20 5,000–45,000

33. Application For determination of the molecular weight of DNA For DNA sequencing To study the purity of DNA To analyze recombinant DNA molecule Can also separate RNA molecule, and its molecular weight can also be determined using calibration curve similar as in DNA

34. D. Pulsed-field gel electrophoresis (PFGE) This technique was developed by Shwartz and Cantor in 1984. Separation of DNA in agarose gel by altering the strength and direction of the electrical field between electrodes. It is used to separate high molecular weight DNA of several mega bases, even whole chromosomes.

35. Application of PFGE PFGE is accurate and results are reproducible with good efficiency, it is used in several areas. Molecular studies of food-borne pathogenic organisms such as Salmonella, E. coli O157:H7, Shigella, Listeria, Campylobacter, etc. The wine industry uses PFGE to monitor the genetic stability of organisms involved in the fermentation processes. PFGE is the first step in cutting and separating large DNA fragments before cloning vectors. It is very useful in mapping applications such as mapping specific disease loci, identifying chromosome rearrangements, RFLP, and DNA fingerprinting. Detecting related strains in case of hospital outbreaks

36. E. SDS-PAGE 1st known as the Laemmli method after its inventor- U.K. Laemmli Upper Stacking gel : It has larger pores with a pH of 6.8 Lower Separating gel: It has smaller pores with a pH of 8

37. Applications To determine the molecular weight of proteins. To fractionate protein subunits To assess the purity of protein samples (because of high resolving power). To determine the isoelectric point(Pi) of a protein. To separate isoenzymes. To fractionate proteins with higher resolution. To study mono, di and tri substituted derivatives of protein. To separate all amphoteric substances.

38. F. 2D electrophoresis The technique of IEF and SDS PAGE combined for fine separation of polypeptides having only minute differences in pI and molecular weight First separation by IEF Next separation according to mol wt (SDS PAGE) which separates protein according to size at right angles to the direction of 1st separation. Series of spots formed in gel which can be quantified, images matched and compared with corresponding spots in related gels

39. G. Immuno-electrophoresis (Rocket Electrophoresis) Principle It combines the sensitivity of gel electrophoresis with the specificity of immune reaction. This technique is used to determine the quantity of a given antigen. Separation Molten agar solution, saturated with a suitable antibody that is complementary against the test antigen to be quantified, is layered on a horizontal plate On the gel, wells are drilled where antigens are filled At the alkaline pH, Ag acquires a negative charge and migrates towards the anode and interact with Ab to form Ag-Ab complex, immunoprecipitates The gel is then stained with CBB, immunoprecipitates will appear in the form of rocket-shaped arcs.

40. The test helps in identification and approximate quantization of various antigens in immunology. suspected monoclonal and polyclonal gammopathies. Used to analyze complex protein mixtures containing different antigens. The medical diagnostic use is of value where certain proteins are suspected of being absent (e.g., hypogammaglobulinemia) or overproduced (e.g., multiple myeloma). This method is useful to monitor antigen and antigen-antibody purity and to identify a single antigen in a mixture of antigens.

41. H. Difference Gel Electrophoresis(DIGE) Up to 3 different protein samples can be labeled with size and charge matched fluorescent dyes (for example Cy3, Cy5, Cy2) the three samples are mixed, loaded and 2D electrophoresis is carried out after which the gel is scanned with the excitation wavelength of each dye one after the other, so we are able to see each sample separately. This technique is used to see changes in protein abundance (for example, between a sample of a healthy person and a sample of a person with disease), post-translational modifications, truncations, and any modification that might change the size or isoelectric point of proteins.

42. Difference Gel Electrophoresis(DIGE) Since the proteins from the different sample types (e.g. healthy/diseased, virulent/non-virulent) are run on the same gel they can be directly compared. To do this with traditional 2D electrophoresis requires large numbers of time-consuming repeats. In experiments comprising several gels, an internal standard in each gel is included. The internal standard is prepared by mixing together several or all of the samples in the experiment. This allows the measurement of the abundance of a protein in each sample relative to the internal standard. Since the amounts of each protein in the internal standard are known to be the same in every gel, this method reduces inter-gel variation

43. Isotachophoresis Used for separation of smaller ionic substances. They migrate adjacent in contact with one another, but not overlapping. The sample is not mixed with the buffer prior to run. Hence current flow is carried entirely by the sample ions. Faster moving ions migrate first and the adjacent ones next with no gap between the zone .

44. All ions migrate at the rate of fastest ion in zones. Then it is measured by UV absorbance. Application Separation of small anions and cations Amino acids Peptides Nucleotides Nucleosides Proteins.

45. Capillary electrophoresis Technique first described by- Jorgensen and Lukas (1980’s) As the name suggest, the separation is carried in a narrow bore Capillary tubes. 25-50μm – Internal diameter 300-330μm – External diameter Length – 50-100cm Fused silica capillary tube Polyimide coating external High voltage is applied (up to 50 kV)

46. Capillary electrophoresis

47. Capillary electrophoresis The components migrate at different rate along the length Although separated by the electrophoretic migration, all the sample is drawn towards cathode by electroendosmosis. Since this flow is strong, the rate of electroendosmotic flow is greater than the Electrophoretic velocity of the analyte ion , regardless of the charge. Positively charged molecule reach the cathode first (electrophoretic migration + electrosmotic flow).

48. Components Reservoirs Electrical buffer Power source Capillary tube Detector and computer Sample applicator

49. Sample application is done by either of one method High voltage injection The buffer reservoir is replaced by the sample reservoir the high voltage is applied buffer reservoir is placed again and voltage applied for the separation . Pressure injection Capillary is removed from buffer and placed in air tight sample with pressure sample is pushed into capillary capillary kept back in the buffer sample and voltage is applied for the separation .

50. Available menu include Serum protein Serum immunotyping Urine immunotyping Multiple myeloma testing Haemoglobinopathy screening HbA1c

51. Detection (Serum protein)

52. Serum electrophoresis Separates proteins into 5 different bands: Albumin, and Prealbumin. Albumin is formed in the liver and is 60% of total proteins. alpha1-globulin ( α1- globulin). alpha 2-globulin ( α2- globulin). beta – globulin ( β- globulin). gamma – globulin ( γ- globulin).

53. Pattern of different bands on serum protein electrophoresis Albumin zone shows only albumin. Alpha1- zone shows : alpha1 – lipoprotein , High-density lipoprotein (HDL) , Alpha-1 -antitrypsin. Alpha 2 zone shows : Alpha-2 macroglobulin , Haptoglobin , β - lipoprotein. Beta zone shows : Transferrin , Complement 3 (C3). Gamma zone shows : Immunoglobulin(IgM, IgA, IgG) .

54. Troubleshooting Adsorption of protein to the wall of capillary – leading to smearing of protein – viewed as peak broadening – or complete loss of protein. - Use of neutral coating group to the inner surface of the capillary.

55. Advantage over conventional Online detection. Improved quantification. Almost complete automation. Reduced analysis time.

56. Microchip electrophoresis Current advanced method. Development in technique include: Integrated microchip design Advanced detection system New application Protein and DNA separation can be done

57. Instrumentation Similar to the capillary electrophoresis. Separation channel Sampleinjection (50-100pL) Reservoirs Voltage (1-4kV) sample preparation Pre column or post column reactors. Classical Cross-T design. Time period of 50-200sec.

58. Detector Laser induced fluorescence Electrochemical detectors Pulsed amperometric detector Sinusoidal voltametry

59. Thank you… Ref.. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 8th Edition Principles and Techniques of Biochemistry and Molecular Biology, Wilson and Walker 8th Edition YouTube and Google images.