THEM6 Antibody / Thioesterase superfamily member 6 (FY13027)

Immunofluorescent staining of THEM6 using anti-THEM6 antibody (green). THEM6 was detected in an immunocytochemical section of SIHA cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-THEM6 antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of THEM6 using anti-THEM6 antibody. Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human RT4 whole cell lysates, Lane 2: human MCF-7 whole cell lysates, Lane 3: human Hacat whole cell lysates, Lane 4: human PC-3 whole cell lysates, Lane 5: rat thymus tissue lysates, Lane 6: rat PC-12 whole cell lysates, Lane 7: mouse thymus tissue lysates, Lane 8: mouse RAW264.7 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-THEM6 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 an ECL Plus Western Blotting Substrate. A distinct band is detected at approximately 20 kDa, slightly below the predicted molecular weight of 24 kDa for full-length THEM6. The faster migration is consistent with literature reports indicating that THEM6 undergoes N-terminal signal-peptide cleavage and contains hydrophobic ER-targeting sequences that reduce SDS binding, producing an apparent size around 19-21 kDa on SDS-PAGE.

Immunoprecipitating THEM6 in PC-3 whole cell lysate. Western blot analysis of THEM6 using anti-THEM6 antibody. Lane 1: PC-3 whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-THEM6 antibody in PC-3 whole cell lysate, Lane 3: anti-THEM6 antibody (2ug) + PC-3 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-THEM6 antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. A distinct band is detected at approximately 20 kDa, slightly below the predicted molecular weight of 24 kDa for full-length THEM6. The faster migration is consistent with literature reports indicating that THEM6 undergoes N-terminal signal-peptide cleavage and contains hydrophobic ER-targeting sequences that reduce SDS binding, producing an apparent size around 19-21 kDa on SDS-PAGE.

Flow Cytometry analysis of MCF-7 cells using anti-THEM6 antibody. Overlay histogram showing MCF-7 cells stained with (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-THEM6 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.