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In this article we will discuss about the principle, requirements and procedure for Polyacrylamide Gel Electrophoresis (PAGE).
Micro-weights of proteins can be separated and characterised rapidly and relatively sensitive, using PAGE. Buffer gels (non-denaturing) as well as Sodium Dodecyl Sulphate (SDS) containing denaturing gels are used. Separation in buffer gels depends on both the charge and size of protein. In SDS gels it depends only on the size.
Polyacrylamide gels are formed by polymerising acrylamide with a cross linking agent bisacrylamide in presence of a catalyst ammonium persulphate and chain inhibitor TEMED. The porosity of the gel is determined by the relative proportion of acrylamide monomer to bisacrylamide. High molecular weight proteins are separated with low percentage gel (large pores) and that for low molecular weight proteins with high percentage gel (small pores).
Polymerisation of the gel is increased by higher concentration of ammonium per sulphate and TEMED.
Principle:
The anionic detergent SDS binds strongly to, and denatures proteins. The number of SDS molecules bound to a polypeptide chain is approximately half the number of amino acid residues in that chain. The SDS-protein complex carries negative charges. Therefore they move towards the anode. Separation is based on the size of protein molecules.
Requirements:
Store in light proof container at 4°C
Adjust pH to 8.8 with concentrated HCI and make up the volume to 200 ml by adding distilled water. Store upto 3 months at 4°C in dark.
Adjust pH to 6.8 with concentrated HCI and adjust the volume to 50 ml with distilled water. Store for 3 months at 4°C in dark.
Store upto 6 months at room temperature.
Use this for subsequent changes of destaining for long term storage of destained gels.
Materials:
1. PAGE vertical system.
2. Shakers.
3. Staining tray.
4. Microsyringes/pipettes.
Procedure:
Assemble the glass plates according to the instructions of the manufacturer and determine the volume of gel mould (information is generally given by the manufacturer).
1. Prepare the volume of solution containing the desired concentration of acrylamide gel (12%) in an Erlenmeyer flask. The various concentrations for preparing resolving gels is given in Table 1. Components are to be mixed in the order shown. As soon as TEMED is added, polymerisation will start. Immediately swirl the mixture rapidly and proceed to the next steps.
[Concentration of ammonium persulphate recommended is higher than that used by some workers. This will eliminate the need to get rid of the dissolved oxygen (which retards polymerization) of the acrylamide solution by degassing].
2. Pour acrylamide solution into the gap between the two glass plates with the meniscus of the acrylamide solution far below the top of the notched plate to allow for the length of the teeth on the comb plus 1 cm.
3. Using a Pasteur pipette carefully overlay the acrylamide solution with water, isopropanol or isobutanol. For gels having concentrations lower than 8% use water; for gels of 10% and above use isobutanol or water saturated isobutanol. This overlaying solution helps in preventing oxygen from diffusing into the gel and inhibit polymerisation of acrylamide gel.
4. Place the gel in a vertical position at room temperature for polymerisation.
5. After the completion of polymerisation (about 30 minutes) pour off the overlay and wash the top of the gel several times with deionised water to remove any unpolymerised gel. Drain as much fluid as possible from the top of the gel. Remove all traces of water from the top of the gel with the edge of paper towel.
6. Prepare stacking gel as given in Table 2. In a small Erlenmeyer flask, prepare appropriate volume of solution containing the desired concentration of acrylamide (Table 2). Mix the components in the given order. As soon as TEMED is added, polymerisation will start and without any delay swirl the mixture rapidly and proceed to next step.
7. Pour the stacking gel solution directly on to the top of the polymerised resolving gel and immediately insert a clean Teflon comb into the stacking gel solution, taking care to see that no air bubble gets in. Add more stacking gel solution to fill the spaces of the comb completely. Place the gel in a vertical position at room temperature (Teflon combs should be cleaned with water and dried with ethanol before use) for 30 minutes for the stacking gel to Set.
Loading of sample:
8. While the stacking gel is setting, prepare the samples for loading. This is done by heating them in boiling water bath for 3 minutes to denature the proteins. This denatured state is maintained by keeping them on ice. A sample containing marker proteins of known molecular weight also is denatured and kept on ice.
9. After completion of polymerisation of the stacking gel (30 minutes) remove the Teflon comb carefully.
10. Wash the wells using a wash bottle with deionised water to remove any unpoly- merised acrylamide. If necessary straighten the teeth of the stacking gel with a long pointed tip.
11. Mount the gel in the electrophoresis apparatus. Add Tris-glycine electrophoresis buffer to the top and bottom reservoiers. Remove any air bubble caught between the plates at the bottom of the gel by squirting, running buffer through a syringe fitted with a bent needle.
12. Load each of the samples in a predetermined order, into the bottom of the wells using a microlitre syringe, or fine disposable tips.
13. Load equal volumes of SDS gel-loading buffer into wells that are unused.
Electrophoresis
14. Attach the electrophoresis apparatus to an electric power supply with positive electrode connected to the bottom buffer reservoier. Apply a voltage of 8V/cm to the gel.
15. After the dye front having been moved into the resolving gel, increase the voltage to 15V/cm and run the gel until bromophenol blue reaches the bottom of the resolving gel. Then put off the power.
16. Remove glass plates from the electrophoresis apparatus and place them on a paper towel.
17. Using a spatula remove the plates apart and mark the orientation of the gel. The gel can now be fixed, stained with Coomassie Brilliant Blue.
Staining of SDS polyacrylamide gel with Coomassie Brilliant Blue:
The polypeptides which are separated by SDS-polyacrylamide gel can simultaneously be fixed with methanol: glacial acetic acid and stained with Coomassie Brilliant Blue R 250. The gel is immersed for several hours in a concentrated methanol acetic acid solution of the dye and then excess dye is allowed to diffuse from the gel during a prolonged period of destaining in dye free methanol/ acetic acid/water.
18. Immerse the gel in atleast five volumes of staining solution and place on a slowly rotating platform for atleast 4 hours at room temperature. The stain can be removed and kept for further use.
19. Destain the gel by soaking it in the methanol acetic acid solution without the dye on a slowly rocking platform for 4— 8 hours, changing the destaining solution three or four times.