Anti-CRKL Antibody Picoband®

Western blot analysis of CRKL using anti-CRKL antibody (A02100-1).
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 30 ug of sample under reducing conditions.
Lane 1: human K562 whole cell lysates,
Lane 2: human 293T whole cell lysates,
Lane 3: human U2OS whole cell lysates,
Lane 4: human Hela 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-CRKL antigen affinity purified polyclonal antibody (Catalog # A02100-1) 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 1:5000 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 CRKL at approximately 35 kDa. The expected band size for CRKL is at 34 kDa.

IF analysis of CRKL using anti-CRKL antibody (A02100-1) and anti-Tubulin Alpha antibody (M03989-3).
CRKL was detected in immunocytochemical section of U87 cell. 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 5 μg/mL rabbit anti-CRKL Antibody (A02100-1) and mouse anti-Tubulin Alpha antibody (M03989-3) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) and DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Immunoprecipitating (IP) CRKL in 293T whole cell lysate.
Western blot analysis of CRKL using anti-CRKL antibody (A02100-1);
Lane 1: 293T whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-CRKL antibody in 293T whole cell lysate;
Lane 3: anti-CRKL antibody (2μg) + 293T whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-CRKL antigen affinity purified polyclonal antibody (A02100-1) at a dilution of 0.5 μg/mL and probed with a mouse anti-rabbit IgG-HRP secondary antibody (Catalog # BM2007). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1196-200). A specific band was detected for CRKL at approximately 34 kDa. The expected band size for CRKL is at 34 kDa.

Flow Cytometry analysis of RT4 cells using anti-CRKL antibody (A02100-1).
Overlay histogram showing RT4 cells stained with A02100-1 (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-CRKL Antibody (A02100-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.