Electrophoresis in biochemistry practical.pptx

Ppt explaining ppt

1. ELECTROPHORESIS

2. ELECTROPHORESIS Movement of charged particles when subjected to an electric field. Positively charged particles ( cations ) move to cathode and negatively charged ones (anions) to anode . By this technique solutes are separated by their different rates of travel through an electric field . Commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids

3. Factors affecting electrophoresis Electrophoresis velocity depends on- Magnitude of its charge Charge density Molecular weight Shape Inherent Factors Solution pH Electric field Solution viscosity Temperature External e n vironmen t -

4. Factors affecting electrophoresis The rate of migration (separation of particles) during electrophoresis depends on : 1. Net charge on the particles (as in case of proteins) 2. The pH of the medium 3. Mass and shape of the particles. 4. Strength of electrical field . 5. Properties of the supporting medium 6. Temperature.

5. 6/22/2014 Biochemistry fo medics- Lecture notes 5 Electrophoretic mobility The Electrophoretic mobility is directly proportional to net charge and inversely proportional to molecular size and viscosity of the electrophoresis medium The p H of solution affects the mobility of the ion by determining the amount and nature of charge Proteins, nucleic acids, nucleotides and amino acids bear charged polar groups making them suitable groups for electrophoresis Carbohydrates carrying no charged groups are first bound to charged groups like Borate or Sulfite ions and then electrophoresis is carried out . Lipids are not electrophoresed because electrophoretic current requires polar solvents in which most lipids are insoluble

6. Types of electrophoresis M ov in g boundary zo n e Ge l Polyacrylamide Non dissociating (Native PAGE) Dissociating (SD S- P A GE) Agarose Paper

7. 6/22/2014 Biochemistry for medics- Lecture notes 7 Types of Electrophoresis Horizontal Vertical Vertical electrophoresis is mainly used for Polyacrylamide gel electrophoresis.

8. 6/22/2014 Biochemistry for medics- Lecture notes 8 Electrophoresis Apparatus Electrophoresis apparatus consists of- Buffer tank -to hold the buffer Buffer Electrodes- made of platinum or carbon Power supply Support media Note-Choice of buffer depends on the nature of substance to be separated and the electricity is supplied at a constant current and voltage.

9. 6/22/2014 Biochemistry for medics- Lecture notes 9 Electrophoresis Apparatus The electrophoresis support on which separation takes place may contact the buffer directly or by means of wicks. The entire apparatus is covered to minimize separation

10. 6/22/2014 Biochemistry for medics- Lecture notes 10 Support media for electrophoresis Filter Paper Cellulose acetate membrane Agar or Agarose gel Starch Gel Polyacrylamide gel

11. Different Support Medium used for Electrophoresis Filter paper : Electrophoresis takes 16–18 hours at a low voltage. Disadvantages Takes a very Long time 2. Diffusion of particles is associated with blurring of margins. Hence no discrete bands are obtained corresponding to different fractions

12. 12 2. Cellulose acetate It is used in the form of cellulose membrane strips. Advantages 1. It takes only one hour for separation of different proteins in a mixture. 2. Separation occurs with formation of discrete bands without any diffusion.

13. 13 Uses of Cellulose acetate membrane Electrophoresis It has been used for separation and identification of different lipoproteins fractions 2. It is also used for separation and identification of different haemoglobins fractions 3. It is also used for separation and identification of different isoenzymes

14. Agarose gel Electrophoresis Procedure of Agarose gel Electrophoresis: Heterogeneous polysaccharides are used. These are viscous liquid when hot but they solidify into a gel on cooling. The gel is prepared in the buffer and spread over slides and allowed to cool . A small sample (few microlitres ) of serum or biological fluid is applied.

15. Electrophoretic run takes around 90 minutes on Agarose gel Electrophoresis.

16. 16 Uses of Agarose gel Electrophoresis It has been used for separation and identification of different Serum proteins fractions 2. It has been used for separation and identification of different protein mixtures. Hence used in different research laboratories 3. It has also been used for separation and identification of different nucleic acids.

17. Electrophoresis apparatus

18. Polyacrylamide gel electrophoresis (PAGE) In this case, support medium is prepared by polymerisation of acrylamide; and creating cross linkages So that finally Microscopic mesh work is formed which acts as support medium .

19. 19 Advantages of PAGE: This type of electrophoresis performs separation of molecules in a mixture using 2 properties simultaneously i.e. Electrical pull; 2. Molecular sieving effect; Hence, Separation is very efficient.

20. In agar gel electrophoresis, serum proteins will show 5 fractions; while in PAGE, serum will show more than 20 different bands

21. Electrophoresis pattern of normal serum sample.

22. High Voltage Electrophoresis In this type of electrophoresis,, higher voltages (400–2,000 volts) are used. Hence called high voltage electrophoresis Since the rate of separation of molecules depends on the strength of the current, the advantage is that the result could be obtained within half an hour.

23. 23 Capillary Electrophoresis Here the gel is taken in a capillary tube of small bore (50–100 microns) with 100–200 cm in length. Nanoliter range of sample is injected into the tube. A high voltage of 25,000 volt is applied. Within a few minutes, components are separated.

24. 24 Uses of Capillary Electrophoresis : It has been used for separation and identification of different Amino acids and Serum proteins fractions 2. It has been used for separation and identification of different drugs 3. It has also been used for separation and identification of different vitamins

25. 4. Also used for separation and identification of different carbohydrates 5.Different nucleotides and nucliec acids can also be separated by this method

26. Working of Capillary electrophoresis

27. Advantages of Capillary electrophoresis 1.Separation time needed is only a few minutes. 2.The quantity of sample required for the separation is in the nanolitre range.

28. After the run, the proteins are fixed to the solid support using acetone or methanol. Then staining is done by ( Amido Schwarz, Ponceau S or Coomassie Blue). Hence identification of different protein fractions is done Visualisation of Protein Bands

29. Scanning of Electrophoretogram Using a Densitometer Light is passed through agar gel plate; Absorption of light will be directly proportional to the quantity of protein present on a band. Hence quantification of different protein fractions is done

30. 1 = Normal pattern; 2 = Multiple myeloma ( M-band) between beta and gamma region.

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32. 33 Major Applications of Electrophoresis It has been used for separation and identification of different seum proteins fractions 2. Different haemoglobins fractions 3. For separation and identification of different isoenzymes

33. 34 Applications of Electrophoresis( Contd ) : 4. It has been used for separation and identification of different drugs 5. It has also been used for separation and identification of different vitamins

34. Further, quantification of different protein fractions can also be very easily done by Scanning of Electrophoretogram Using a Densitometer So in nutshell, Electrophoresis helps in very precise identification as well as quantification of different closely related fractions in a mixture

35. Electrophoresis pattern of normal serum sample.

36. 1 = Normal pattern; 3 = Chronic infection, broad based increase in gamma region; general increase in alpha1 and alpha2 bands;

37. 1 = Normal pattern; 2 = Multiple myeloma ( M-band) between beta and gamma region.

38. 1 = Normal pattern; 4 = Nephrotic syndrome; hypoalbuminemia ; prominent alpha-2 band

39. 1 = Normal pattern; 5 = Cirrhosis of liver; decreased albumin band

40. Normal Albumin level in blood is 3.5 - 5 g /dl Normal Albumin : Globulin Ratio is 1.7:1 to 2:1

41. Causes of Reversal of Albumin : Globulin Ratio This can be either due to: Clinical diseases associated with reduction of albumin Many Clinical conditions associated with increase in globulin fractions

42. Causes of Hypoalbuminemia 1.Cirrhosis 2.Nephrotic syndrome 3.Malnutrition 4.Malabsorption syndromes

43. 1.Reduction of albumin(Compensatory increase) 2.Chronic infections (gamma fraction) 3.Multiple myeloma (gamma fraction ) 4. HyperLipoproteinemias (beta fraction ) 5. Nephrotic syndrome (alpha fraction ) Causes of Hyper Globulinemias

44. After the run, the proteins are fixed to the solid support using acetone or methanol. Then staining is done by ( Amido Schwarz, Ponceau S or Coomassie Blue). Hence identification of different protein fractions is done Visualisation of Protein Bands

45. 3. It is also used for separation and identification of different isoenzymes e.g. isoenzymes of CK, LDH & ALP Hence it is helpful in diagnosis of: MI Muscular dystrophies & Differentiating b/w physiological and pathological causes of rise in ALP

46. Further, quantification of different protein fractions or different isoenzymes can also be very easily done by Scanning of Electrophoretogram Using a Densitometer So in nutshell, Electrophoresis helps in very precise identification as well as quantification of different closely related fractions in a mixture

47. 49 One of the major Applications of Electrophoresis include separation and identification of different haemoglobins fractions. Hence useful in diagnosis of many haemoglobinopathies .

48. Haemoglobin Electrophoresis The different closely related fractions of haemoglobin in a mixture can be a mixture of different haemoglobin variants in case of haemoglobin Electrophoresis.

49. It is used for diagnosis of many different haemoglobinopathies , as well as to study normal haemoglobin variants in a particular population. E.g. HbD is a common normal haemoglobin variants in Punjabi population.

50. The important haemoglobinopathies which are diagnosed with haemoglobin Electrophoresis include : Thalassemia major and minor (associated with increase in Hb F and a slight increase in HbA2 fraction)

51. 2. Sickle cell Anaemia which is associated with abnormal Hb S band between Hb A and Hb F band Thus Electrophoresis makes vast use in confirmation of clinical diagnosis as well as for research purpose

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