6 Matching Annotations
  1. May 2019
    1. Expression conditions for bZP3 under the polyhedrin promoter were standardized using the Northern blot and Western blot analysis of cells infected with the VI virus. Sf9 cells, seeded at a density of 1.5 million in a 35 mm petridish were allowed to attach for I h at 27oc. The medium was removed and the cells were infected with AcNPV (Autographa californica nuclear polyhedrosis virus) or VI at -10 MOl for 1 h. The infected cells were harvested at different time points from 0-84 h pi. The cells (-2X I o6) were washed with chilled PBS and resuspended in I ml of denaturing solution ( 4 M GITC, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, and 0.1 M BME) followed by addition of 50 Jll of 2 M sodium acetate (pH 4) and 500 Jll water saturated phenol and 1 00 Jll chloroform:isoamyl alcohol ( 49: 1 ). The suspension was mixed thoroughly after the addition of each reagent, vortexed for 1 0 sec and cooled on ice for 15 min. The aqueous and the phenol phases were separated by centrifugation at 12,000 rpm for 20 min in a refrigerated microfuge. The aqueous phase was transferred to a fresh tube and 500 Jll isopropanol was added. RNA was precipitated at -20°C for 1 h, and pelleted at 12,000 rpm for 20 min at 40C. The RNA pellet was dissolved in 300 Jll denaturing solution followed by addition of 300 Jll of isopropanol. RNA was reprecipitated at -2ooc for 1 h, washed with 75% ethanol and the pellet collected by centrifugation at 12,000 rpm in a refrigerated microfuge. RNA was dissolved in 25 Jll of 0.5% SDS by heating at 65°C for 10 min and stored at -700C. RNA was quantitated and 5 Jlg of RNA corresponding to each time point was resolved on a 1.2% agarose formaldehyde gel, transferred to a nylon membrane and probed with 32p labeled bZP3 probe. Cells harvested at different time points from -2X 106 cells 12-84 h pi were pelleted down, washed with 10 mM PBS, pH 7.4, and lysed in reducing buffer and resolved on a 0.1% SDS-10% PAGE as described earlier. The supernatant was concentrated to lOX for loading on the gel.
    2. Expression 'Of bZP3 in BEVS
    1. Viewing slides under microscope
    2. A drop of immersion oil was put on top of the cover-slip before viewing it under microscope. The cells were viewed at 100X resolution of Nikon Eclipse 80i microscope.Thedifferential interference contrast images of the cells were captured using NIS-Elements D3.0 software also used to find out mean cell size using at least 100 randomly selected cells.Fluorescence images were captured on Zeiss LSM 710 Meta inverted confocal microscope
    3. Band intensities in gel autoradiogramswere determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system. Equal areas of radioactive bands were boxed and the PSL (Photo stimulated luminescence) values were further considered. Background signal (obtained from equal area as that of the radioactive band but from other part of the gel/blot) is subtracted from the signal intensities obtained from radioactive bands to get the final values
    4. Densitometry