5.2.9 Methods
The prepared plant sample and each standard were injected in HPLC and the
following running program was followed:
Wavelength
280 nm
Flow rate
1.0 mL/min
Total run time 25 min
Solvent A
Methanol
Solvent B
1% formic acid in water (Lin et al. 1996)
The gradient program was started with 60% of solvent A and obtaining peak was
changed into 35, 10, and 60% of elute A at retention times 5, 10, 15, and 20 min,
respectively (Boligon et al. 2012).
5.2.10 Protein Extraction
Total proteins were extracted by adding l ml of 50 mM Tris-HCl (pH 7.5) and 0.5 M
NaCl at 4
C to 30 mg of fresh radish for 60 min. Then it was frozen at À20
C and
thawed three times for 24 h to disrupt the tissue and to liberate the proteins (Miller
1972). It was centrifuged at 10000 rpm for 15 min.
5.2.11 SDS-PAGE Electrophoresis
Protein was extracted through SDS-PAGE (Laemmli 1970). 10 μL of extracted
protein was mixed with 2.5 μL of 2-mercaptoethanol and 7.5 μL of 0.002%
bromophenol blue in 0.0625 M Tris-HC1 (pH 6.8), containing 10% glycerol and
2% SDS, and was kept into SDS-PAGE gel apparatus (Medox). All protein staining
was performed using Coomassie blue (Hames 1998).
5.3 Results and Discussion
5.3.1 Morphological Characteristics of Organically
and Inorganically Grown Plant
Table 5.1 shows the physicochemical characteristics and heavy metal content of the
soil. Growth parameters denote that the organically grown Raphanus sativus
5 Influence of Distillery Sludge-Based Vermicompost on the Nutritional Status of. . .
73
The prepared plant sample and each standard were injected in HPLC and the
following running program was followed:
Wavelength
280 nm
Flow rate
1.0 mL/min
Total run time 25 min
Solvent A
Methanol
Solvent B
1% formic acid in water (Lin et al. 1996)
The gradient program was started with 60% of solvent A and obtaining peak was
changed into 35, 10, and 60% of elute A at retention times 5, 10, 15, and 20 min,
respectively (Boligon et al. 2012).
5.2.10 Protein Extraction
Total proteins were extracted by adding l ml of 50 mM Tris-HCl (pH 7.5) and 0.5 M
NaCl at 4
C to 30 mg of fresh radish for 60 min. Then it was frozen at À20
C and
thawed three times for 24 h to disrupt the tissue and to liberate the proteins (Miller
1972). It was centrifuged at 10000 rpm for 15 min.
5.2.11 SDS-PAGE Electrophoresis
Protein was extracted through SDS-PAGE (Laemmli 1970). 10 μL of extracted
protein was mixed with 2.5 μL of 2-mercaptoethanol and 7.5 μL of 0.002%
bromophenol blue in 0.0625 M Tris-HC1 (pH 6.8), containing 10% glycerol and
2% SDS, and was kept into SDS-PAGE gel apparatus (Medox). All protein staining
was performed using Coomassie blue (Hames 1998).
5.3 Results and Discussion
5.3.1 Morphological Characteristics of Organically
and Inorganically Grown Plant
Table 5.1 shows the physicochemical characteristics and heavy metal content of the
soil. Growth parameters denote that the organically grown Raphanus sativus
5 Influence of Distillery Sludge-Based Vermicompost on the Nutritional Status of. . .
73