1. PAGE – SLAB GEL ELECTROPHORESIS

2. Introduction  Electrophoresis is defined as the migration of the charged particles or molecules in solution under the influence of electric field.  It is used to separate mixture of amino acids, proteins and Nucleic acids.  Electrophoretic mobility (µ)= Velocity of migration (V) Electric field strength (E)

3. Types 1) Paper Electrophoresis 2) Agarose gel Electrophoresis (AGE) 3) Polyacrylamide gel Electrophoresis (PAGE) 4) Immunoelectrophoresis

4. Polyacrylamide gel Electrophoresis (PAGE) Polyacrlamide gel - synthetic gel PAGE is used in the separation of wide range of molecules even in extreme experimental conditions like pH, Temperature and electrical conditions. PAGE is carried out in glass tubes – DISC or Tube electrophoresis. Recently, a more convenient method called Slab gel electrophoresis is used.

5. Procedure 1. Assemble glass plates for preparing a slab as shown in the pictures.

6. 2. Place a thoroughly cleaned rectangular plate on a table and keep three gaskets. 3. Place the notched glass plate over the gaskets and insert two spacers along the gaskets. 4. Using metal clips, clamp together the rectangular and notched glass plate assembly. 5. Keep the whole assembly in a vertical position by placing them in a gel casting stand. Tighten it gently.

7. 6. Prepare separating gel mixture with the following; Acrylamide stock solution – 10 ml 4X separating gel buffer – 7.5 ml 10% Sodium dodecyl sulphate - 0.3 ml Distilled water - 12.1ml 10% Ammonium per sulphate- 150 µl TEMED- 10 µl

8. 7. Using a 10 ml pipet, pour the gel mixture immediately into the glass sandwich up to the mark and allow no air bubbles. 8. Immediately after pouring the separating gel mixture, take about 0.5-1.0 ml of n-Butanol saturated with buffer or water or separating gel overlay solution and inject it slowly. The overlay solution spreads over the entire top surface of the separating gel mixture. 9. Allow the gel undisturbed for polymerization for about 15-20 min.

9. 10. After polymerization, tilt the sandwich and discard the overlay solution and wash the surface with separating gel overlay. 11. Preparation of stacking gel mixture; – Acrylamide stock- 1.33 ml – 4% stacking gel buffer- 2.5 ml – 10% SDS solution- 0.1 ml – Distilled water- 6.0 ml – 10% APS- 50 µl – TEMED- 5 µl

10. 12. Discard the overlay solution, insert the comb and pour the stacking gel mixture. 13. Allow the polymerization to complete for about 15-30 min. In the mean time prepare samples. 14. Carefully remove the comb, rinse the wells with tank buffer and remove the bottom. 15. Clamp the gel plate and tighten it. 16. Using micro pipette, apply 10 µl of sample to each well.

11. 17. Slowly fill up the top chamber with tank buffer and fill the wells with buffer very gently using a syringe. 18. Add equal volume of tank buffer to the bottom reservoir. 19. Connect the tank to the power supply and turn the power on. Set the power supply to constant current mode. Apply 10-15 mA current.

12. 20. Continue the run, till the marker dye reaches the bottom of the gel. 21. Decrease the power and turn off the power supply. Disconnect the power cord. 22. Drain the upper reservoir buffer and take the gel out and carefully remove the top notched plate. 23. Place the gel in dye solution, pour enough dye solution to cover the gel and stain it for 3- 4 hours.

13. 24. Remove the staining solution and add destain. Shake it intermittently and change the destain several times until a clear background is obtained. 25. Examine the bands in a white light transilluminator.

16. 1. Choose the correct sequence of steps involved in Polyacrylamide gel Electrophoresis I) Staining of the gel II) Loading of separating gel III) Application of sample IV) Loading of stacking gel a) II, IV, III, Ib) I, II, III, IV c) III, II, IV, I d) I, IV, II, III 2. The commonly used stain in Electrophoresis is a)Methylene blue b)Coomassie brilliant blue c)Methyl orange d) Haematoxylin

17. 3. Of the following pair of statements one is an assertion and the second is a possible reason.Read the statements carefully and mark your answer according to the keys given below. Assertion: Mixture of biological samples can be separated by Electrophoresis. Reason: In Electrophoresis, smaller molecules move faster than the larger one.

18. a) Both the Assertion and Reason are true statements and the Reason is an adequate explanation for the Assertion. b) Both the Assertion and Reason are true statements but the Reason does not explain the Assertion. c) Assertion is a true statement, and the Reason is a false statement. d) Both Assertion and Reason are false statements. 4. The electrophoretic technique that involves use of two gel systems is a) AGE b) PAGE c) Paper d)Isoelectrofocussing