Anti turbogfp antibody

To understand the subcellular localization of large yellow croaker RIP3, HEK 293T cells were seeded on sterilized coverslips in 6-well plates at a density of 2 × 10⁵ cells per well. The following day, the cells were transfected with 5 μg of plasmid constructs of pTurbo-RIP3-GFP or pTurboGFP-N (vector control) using Lipofectamine 3000 according to the manufacturer’s instructions. At 24 h post-transfection, the cells were washed with 1× PBS, fixed using 4% paraformaldehyde, and permeabilized with Triton X-100 (0.2% in PBS) for 15 min at room temperature, respectively. The coverslips were then washed with PBS and overlaid with one drop of the mounting medium (VECTASHIELDR Hard Set™ Mounting Medium with DAPI, Vector Laboratories, CA, USA). After staining with DAPI, the cells were visualized and imaged using a confocal microscope (Leica TCS SP8, Germany). The cells were subsequently collected and lysed in a RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors (Beyotime, Shanghai, China) for the detection of RIP3-GFP and the pTurboGFP fusion proteins using a Western blotting analysis as per our previous reports [20 (link),24 (link)] with the primary antibody (Anti-TurboGFP antibody, Evrogen, CAT. #AB513) diluted at 1:5000.

PCR product was ligated into multiple cloning sites of pCMV-AC-GFP after digestion of restriction enzymes, Sgf I and Mlu I. pCMV-AC-GFP was purchased from ORIGENE (Catalog #PS100010), Sgf I enzyme from NEB (Catalog #R0630S), Mlu I enzyme from NEB (Catalog #R0198S), and T4 ligase from Promega (Catalog #M180A). We transfected HEK293 cells with constructed plasmid 48 h before collecting cells in lysis buffer. Western blot experiments were conducted using these cell lysates. Anti-TurboGFP antibody was purchased from Evrogen (Catalog #AB513).

Transduced HEK-EF1-Luc2-T2A-copGFP or HEK-EF1-CBR2opt-T2A-copGFP were seeded in a 96-well black plate at a density of 50,000 cells/well, left to adhere, washed and fixed using 3.7% formaldehyde in PBS for 15 min, and then treated with 0.1% saponinin PBS for 10 min at ambient temperature. Wells were rinsed three times for 5 min each with ambient temperature PBS (100 µL per well). Cells were then blocked in Blocking Buffer (LI-COR Biosciences) for 1 h at ambient temperature (50 µL per well). Blocking Buffer was removed and then buffer (negative control) or 1:1,000 rabbit polyclonal anti-TurboGFP antibody (Evrogen AB513) was added to the wells (total volume 50 µL per well). The plate was covered and incubated overnight at 4 °C. The next day, cells were washed 3× in PBS and then anti-Rabbit IgG (H+L) (DyLight® 800 Conjugate) secondary antibody (diluted in Antibody Dilution Buffer (LI-COR Biosciences), (total volume 50 µL per well)) was added. After a 1 h incubation at ambient temperature in the dark, cells were washed 3× with PBS and scanned using an Odyssey scanner (LI-COR Biosciences) using the following settings: filter 800, intensity 5, and resolution 42 µm.