NSUN6 Antibody / NOP2/Sun RNA methyltransferase family member 6 (FY12291)

Immunohistochemical staining of NSUN6 using anti-NSUN6 antibody. NSUN6 was detected in a paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-NSUN6 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Western blot analysis of NSUN6 using anti-NSUN6 antibody. Lane 1: human 293T whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: human Jurkat whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: rat PC-12 whole cell lysates, Lane 7: mouse brain tissue lysates, Lane 8: mouse Neuro-2a whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NSUN6 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. Expected molecular weight of NSUN6 ~52 kDa (469 aa). Observed band at ~55 kDa, consistent with published data showing NSUN6 migrating slightly above its predicted size and across antibody-validation sources (52-55 kDa). The ~3 kDa upward shift may reflect minor post-translational modification or gel-migration behavior rather than an alternate isoform.

Flow Cytometry analysis of 293T cells using anti-NSUN6 antibody. Overlay histogram showing 293T cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-NSUN6 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.