SDS PAGE

9. SDS PAGE

This is a useful method to separate and/or identify proteins and nucleic acids, based on the polypeptide length to determine their molecular weight. The procedures used in the experiments were based on the Laemmli discontinuous gels. Initial steps involved the preparation of crude extracts from plant leaves (namely egg plant and tomato) where fresh leaves were chopped into small pieces and ground into fine powder with liquid nitrogen. The powder was melted, centrifuged at 15000 rpm for 15 minutes and supernatant transferred to a new tube and stored in ice.

The sample was prepared for SDS page by adding 15 µl of 4 x SDS buffer into a micro tube and 45 µl of crude extract, mixed well and mixture heated at 95 degrees C for 5 minutes.

The separating gel was prepared by assembling the glass plates and the acrylamide solution prepared in a 15 ml falcon tube, and mixed well by inversion. The solution was poured slowly into the spaces between the glass plates, ensuring that all bubbles were removed (if any), and a layer of small amount of water-saturated 2-butanol added carefully onto the surface of the gel solution, to prevent the inhibitory effect of oxygen for polymerization. The glass was subsequently covered with saran wrap to prevent drying.

Following steps included the removal of the overlayed butanol and using kimwipe to soak as much water as possible to by capillary action. The acrylamide solution for stacking the gel was prepared and mixed well by inversion, and then a clean comb was inserted into the gap of the plates thus making sample lanes. It was vital to avoid any air bubbles being trapped under the comb.

To run the electrophoresis, all the SDS-PAGE apparatus were assembled appropriately, comb carefully removed and the samples applied into the desired wells. The samples were ran at 15mA per gel for 90 minutes.

CBB staining was done to detect the proteins in the PAGE gel. In this experiment, a commercially available tool kit (ULTRA-FAST Coomassie Stain) was adopted. The gel was washed twice in 100 ml deionized water, and an adequate amount of staining solution added, and was shaken gently for 30 to 60 minutes. It was finally rinsed in large volumes of deionized water to enhance the intensity of protein bands.