Chilo partellus male moths were collected from the maize fields by using pheromone traps in the farmer fields in Vaddeswaram, Guntur, India.
Sample protein preparation
Male C. partellus moth antenna tissue (100 mg) was taken and homogenized with the help of mortar and pestle using 500 μl of 1 M Tris buffer (pH 7.5) containing 10% SDS and 0.02 M EDTA. The homogenate was centrifuged at 14,000 rpm for 12 min at 4 °C. Supernatant fraction was carefully collected in fresh autoclaved tubes; the sample was stored at − 20 °C until further use. The protein was estimated according to standard protocol suggested by Lowry et al. (1951) using bovine serum albumin (BSA) as standard.
Preparation of sample protein for SDS-PAGE
A ratio of 1:1 protein sample and 1.0 M Tris buffer (pH 6.8) containing 10% SDS, 2% β-mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue was added and kept at 90 °C for 5 min (Kasim et al. 2015).
Separation of PBPs by SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular weight distribution of insect proteins as described by Nagnan-Le et al. (1996). To study the SDS-PAGE protein profile, one-dimensional SDS-PAGE (12% separating gel and 4% stacking gel) was carried out in a small vertical system. To 10 μg of protein, 10 μl of lamellae buffer (loading dye) was mixed and samples were incubated at 95 °C for 5 min in water bath and then immediately load the protein sample into the wells. The loading buffer containing bromophenol blue acts as a tracking dye. A medium range marker (NEX-GEN blue spray prestain protein ladder) was also loaded into the well for the determination of molecular weight through protein bands. The gel was run at constant voltage of 80 V for 3–4 h followed by staining with Coomassie brilliant blue R-250 overnight. The relative mobility of protein bands was determined and the zymogram was constructed.
Extraction and purification of proteins from SDS-PAGE
After SDS-PAGE electrophoresis, proteins were extracted and purified according to Nagnan-Le et al. (1996). The visualized bands are removed from the gel using a surgical blade and washed using 2 ml of 250 mM Tris buffer/250 mM EDTA, pH of 7.4, thoroughly for three times then finally washed with autoclaved distilled water. The water was completely removed from the gel slices and homogenized with 1 ml of Tris buffer (pH 7.4). The samples were subjected to sonication then centrifuged at 12,000 rpm for 10 min at 4 °C. The protein sample was equilibrated with 20 mM Tris buffer, pH 7.4, containing 0.1% SDS. The partially purified protein aliquots were run in the native PAGE; it is used for the densitometric analysis and determination of the recovery percentage.
To identify the universal expression pattern of existing protein spots on gel, densitometry analysis was employed. It is majorly used to measure protein expression levels and the intensities of specific bands by using an online software tool (Gelquant.NET). In this study, the relative amounts of protein were quantified by using a computational program from a TIFF image assisted with a commercial scanner. Relative mobility (Rf) of each stained band, its concentration, and area occupied by it with reference to total protein present in the lane can be calculated by gel perfect program explained by Bozzo and Retamal (1991). SDS-PAGE gels of purified proteins were digitized, and peak areas of both purified and crude protein bands were compared. The relative concentration of areas provides us the percentage recovery.