RAD54L2 Antibody / RAD54-like 2 (FY13030)

Immunofluorescent staining of RAD54L2 using anti-RAD54L2 antibody (red) and anti-Beta Tubulin antibody (green). RAD54L2 was detected in immunocytochemical section of cell. 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-RAD54L2 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. Cy3 Conjugated Goat Anti-Rabbit IgG and DyLight 488 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of RAD54L2 using anti-RAD54L2 antibody. Lane 1: human Jurkat whole cell lysates, Lane 2: human HEL whole cell lysates, Lane 3: human SH-SY5Y whole cell lysates, Lane 4: rat PC-12 whole cell lysates, Lane 5: mouse Raw264.7 whole cell lysates, Lane 6: mouse Ana-1 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-RAD54L2 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. A single prominent band is detected at ~190-200 kDa, higher than the predicted molecular weight of 163 kDa. This apparent shift is consistent with published observations for RAD54L2 and is attributed to its coiled-coil structure and post-translational modifications, including phosphorylation and sumoylation, which collectively cause slower migration on SDS-PAGE.

Flow Cytometry analysis of HEL cells using anti-RAD54L2 antibody. Overlay histogram showing HEL 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-RAD54L2 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 (Red line) was also used as a control.