PUF60 Antibody / Poly(U)-binding splicing factor 60 kDa (FY13163)

Immunohistochemical staining of PUF60 using anti-PUF60 antibody. PUF60 was detected in a paraffin-embedded section of human breast 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-PUF60 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 PUF60 using anti-PUF60 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human COLO-320 whole cell lysates, Lane 2: human SW620 whole cell lysates, Lane 3: human Caco-2 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-PUF60 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. The expected molecular weight of PUF60 is ~60 kDa.

Immunohistochemical staining of PUF60 using anti-PUF60 antibody. PUF60 was detected in a paraffin-embedded section of human breast 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-PUF60 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.

Flow Cytometry analysis of Caco-2 cells using anti-PUF60 antibody. Overlay histogram showing Caco-2 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-PUF60 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.

Immunoprecipitating (IP) PUF60 in MCF-7 whole cell lysate. Western blot analysis of PUF60 using anti-PUF60 antibody; Lane 1: MCF-7 whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-PUF60 antibody in MCF-7 whole cell lysate; Lane 3: anti-PUF60 antibody (2ug) + MCF-7 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-PUF60 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. The expected molecular weight of PUF60 is at 60 kDa.