CBL Recombinant Rabbit Monoclonal Antibody [PSH17-61]

Western blot analysis of CBL on different lysates with Rabbit anti-CBL antibody (HA723936) at 1/5,000 dilution.

Lane 1: Raji cell lysate (20 µg/Lane)
Lane 2: K-562 cell lysate (20 µg/Lane)
Lane 3: COS-1 cell lysate (20 µg/Lane)
Lane 4: Mouse testis tissue lysate (30 µg/Lane)
Lane 5: Rat testis tissue lysate (30 µg/Lane)

Predicted band size: 100 kDa
Observed band size: 110 kDa

Exposure time: 20 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723936) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of Raji cells labeling CBL with Rabbit anti-CBL antibody (HA723936) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CBL antibody (HA723936) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of Raji cells labeling CBL.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA723936, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

CBL was immunoprecipitated from 0.2 mg Raji cell lysate with HA723936 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723936 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: Raji cell lysate (input)
Lane 2: HA723936 IP in Raji cell lysate
Lane 3: Rabbit IgG instead of HA723936 in Raji cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 10 seconds; ECL: K1801