Anti-ROBO1 Antibody Picoband™

Figure 1. Western blot analysis of ROBO1 using anti-ROBO1 antibody (A01530-2).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human U-87MG whole cell lysates
Lane 2: human HEK293 whole cell lysates
Lane 3: human T-47D whole cell lysates
Lane 4: human HepG2 whole cell lysates
Lane 5: human A549 whole cell lysates
Lane 6: human PC-3 whole cell lysates
Lane 7: human Hela whole cell lysates
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-ROBO1 antigen affinity purified polyclonal antibody (Catalog # A01530-2) at 0.5 μg/mL overnight at 4°C, 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 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for ROBO1 at approximately 250KD. The expected band size for ROBO1 is at 181KD.

Figure 2. IF analysis of ROBO1 using anti-ROBO1 antibody (A01530-2).
ROBO1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-ROBO1 Antibody (A01530-2) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG (BA1142) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Figure 3. Flow Cytometry analysis of PC-3 cells using anti- ROBO1 antibody (A01530-2).
Overlay histogram showing PC-3 cells stained with A01530-2 (Blue line).The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti- ROBO1 Antibody (A01530-2, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.