Southern Bolot Hybridization Analysis: Southern Blot Hybridization

3.6.5. Southern hybridization
Southern blot hybridization analysis was carried out using standard procedures (Sambrook et al. 2001). Approximately 20 µg of total genomic DNA from transgenic plants and untransformed control plant was digested with EcoRI, and then separated by electrophoresis on 0.8% agarose gel. Digestion profile was checked with 1µg DNA, with single cutter enzymes EcoRI (10U) in T-DNA region. For southern experiment, 20µg DNA was digested overnight. Restricted fragments were separated on 0.8% agarose gel at 40V for 8 h. De-purination (0.25N HCl) was done in glass pot for 15 min with shaking at room temperature. Subsequently rinse the gel in autoclaved distill water two times for 10 min interval at room temperature (RT). Further
After exposure, return to the darkroom and close the door wear latex gloves than turn on the safelight and open the cassette. Place the film in the developer for approximately 30 sec with periodic agitation, transfer the developed film to water to remove excess developer and agitate for 2 to 3 min further place the film in fixer and leave for 2 to 3 min.
3.6.9. Expression analysis of recSRL by hemagglutination assay
Putative transformants T1 lines (G1, G2, G3, G4 and G5) maintained in greenhouse were used for extraction of soluble protein. Lectin was extracted from leaves (2g) in 2 ml of TBS containing 0.25mM isopropylthio-ß-D-galactoside (IPTG; Merck) for overnight at 4 °C, the homogenate was centrifuged at 6700g for 20 min at 4° C. The supernatant collected was used for hemagglutination assay. The concentration of total soluble protein was determined by the Bradford method (1976).Further the quantity of recSRL expressed in transgenic cotton plants were determined by enzyme linked immunosorbent assay (ELISA).
3.6.10. Western
1988). The total soluble proteins (TSP) extracted (as mentioned above) were heat treated at 50 °C for 30 m to concentrate lectin. Heat labile host proteins precipitated and were separated by centrifugation at 6700 g for 20 min at 4 °C. After heat treatment, protein content was estimated and the concentration was adjusted to ~40 µg. Concentrated lectin in the leaf sample was separated by SDS-PAGE (12% gel) according to Laemmli 1970, the fractionated proteins were transferred onto PVDF membrane (0.45mm; Millipore USA). After blocking for nonspecific binding using 3% BSA, the membrane was incubated with rabbit anti-SRL polyclonal antibodies at 1:5,000 dilutions followed by treatment with a secondary antibody, horse radish peroxidase (HP) conjugated anti-rabbit IgG at 1:2,000 dilutions (Biorad USA). Bound secondary antibodies were detected by Immuno-StarTm HP reagent (Biorad USA) and the blots were exposed to X-ray film followed by