Schematic of the modified zymogram. a: add the mix of loading buffer and crude enzyme solution to the gel. But the mix was not heated at 100°C. Blue: crude enzyme from glucose based medium; Green: crude enzyme from corn stover + wheat bran based medium; Red: loading buffer only. b: after electrophoresis, the first column of each sample was cut out. c: the first column of each sample was stained with CBB. d: the remaining gel was soaked in Tris–HCl buffer to remove SDS. e: the first column after CBB staining was used as a marker to cut the protein bank of the second column. f: the protein bank cut of the second column was soaked in p NPG for active staining. g: after adding Na2CO3, the band coming from the second column kept colorlessness. h: the color of the band from the second column changed from colorlessness to yellow following addition of Na2CO3. i: according to the position of active band of the second column, cut the corresponding bands of the third and fourth column. j: protein coming from the bands of the third and fourth column was injected to the gel for SDS-PAGE following a series of treatments.