SPT6 Antibody / SUPT6H / Transcription elongation factor SPT6 (FY13391)

Western blot analysis of SUPT6H using anti-SPT6 antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: monkey COS-7 whole cell lysates, Lane 2: rat testis tissue lysates, Lane 3: rat C6 whole cell lysates, Lane 4: mouse testis tissue lysates, Lane 5: mouse NIH/3T3 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-SUPT6H 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 is developed using an ECL Plus Western Blotting Substratewith Tanon 5200 system. A strong band was detected at approximately 250 kDa, which is higher than the predicted 199 kDa and consistent with the known slow-migrating behavior of SUPT6H caused by its acidic, low-complexity domains and extensive phosphorylation.

Western blot analysis of SUPT6H using anti-SPT6 antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human HEL whole cell lysates, Lane 2: human whole cell lysates, Lane 3: human SiHa whole cell lysates, Lane 4: human MCF-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-SUPT6H 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 is developed using an ECL Plus Western Blotting Substratewith Tanon 5200 system. A strong band was detected at approximately 250 kDa, which is higher than the predicted 199 kDa and consistent with the known slow-migrating behavior of SUPT6H caused by its acidic, low-complexity domains and extensive phosphorylation.

Immunofluorescent staining of SUPT6H using anti-SPT6 antibody (green). SUPT6H was detected in an immunocytochemical section of human TPC1 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-SUPT6H antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37oC. The tissue section was developed using Phalloidin-iFluor 594 conjugate (red). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of human SiHa cells using anti-SPT6 antibody. Overlay histogram showing SiHa 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-SUPT6H 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.