V5 Tag Antibodies

Immunofluorescence analysis of V5 tag was done on HEK-293 cells transiently overexpressing V5-His/LacZ. Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with V5 Tag Monoclonal Antibody (2F11F7) (Cat. No. 377500) at a dilution of 1:500 in 1% BSA and incubated for 3 hours at room temperature followed by labeling with Rabbit anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Cat. No. A11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Cat. No. A12381). Panel d is a merged image showing cytoplasmic localization. Panel e is untransfected HEK-293 cells.

Flow cytometry analysis of V5 tag was done on HEK-293 cells transiently overexpressing V5-His-LacZ. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with V5 Tag Monoclonal Antibody (2F11F7) (Cat. No. 377500, red histogram) or with mouse isotype control (pink histogram) at a dilution of 1:250 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Rabbit anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Cat. No. A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Western blot analysis of V5 tag. Analysis was performed by loading 25 µg of 293T cells transfected with 12Tag onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4°C overnight. The membrane was probed with a V5 Tag Polyclonal Antibody (Cat. No. PA1-993) at a dilution of 1:2,000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hour at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Cat. No. 32132). Results show a band at ~45 kDa.