To 5.0 ml of 10% acrylamide solution, 2.5 ml of double distilled water and 2.5 ml of Tris–HCl buffer (pH 8.5) was added. Enzyme was added to the reaction mixture and incubated at 45 C for 30 min. After incubation, 200 ll of 10% ammonium persulphate and 20 ll of tetramethylethylenediamine were added. Tubes were kept at room temperature and time of solidification was noted.