Mammalian lysis buffer

HEK293T cells were seeded in 6-well plate (6×10⁵ cells/well) and transfected with 1 μg of plasmids expressing flaghBST2 using Fugene® HD (Promega). At 24 hours post-transfection (hpt), cleared cell lysates were collected by centrifuging at 10,000 rpm for 5 mins and lysed with 100 μl of mammalian lysis buffer (Thermofisher Scientific) supplemented with protease inhibitor cocktail (Thermofisher Scientific). The samples were treated with PNGase F (NEB) following the manufacturers’ instruction with slight modifications. Briefly, 25 μl of sample was mixed with 1X denaturing buffer in a 30 μl total reaction volume. The mixture was boiled for 10 mins and chilled on ice for 1 min. Four microliters of Glycobuffer 2, 4 μl of 10% NP-40, 1 μl of PNGase F and 1 μl of distilled water were then added making a 40 μl total reaction volume. The reaction was incubated at 37°C overnight. The extent of deglycosylation of hBST2 in treated samples was examined by mobility shifts on SDS-PAGE gel. Western blot analysis was performed to visualize hBST2 on nitrocellulose membrane using rabbit α-flag and goat α-rabbit IgG HRP antibodies.

Cells were harvested using ACCUTASE™. Cell pellets were then lysed with mammalian lysis buffer (Thermo Fisher Scientific) containing 1Χ Halt™ protease inhibitor cocktail (Thermo Fisher Scientific). Cleared lysates were subjected to SDS-PAGE. Separated proteins on acrylamide gels were transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk for 1 h and probed with a primary antibody diluted in 5% skim milk for 1 h. Next, the membranes were washed three times with Tris-based saline and 0.1% Tween-20 (TBST) and probed with a secondary antibody diluted in 5% skim milk for 1 h. After the washing step, the protein bands were detected by adding chemiluminescence substrate (Bio-Rad) and visualized using ChemiDoc™ XRS+ imager (Bio-Rad).