CD134 Recombinant Rabbit Monoclonal Antibody [PSH10-14]

☑ Relative expression (RE)

Western blot analysis of CD134 on different lysates with Rabbit anti-CD134 antibody (HA723183) at 1/2,000 dilution.

Lane 1: HUT 102 cell lysate
Lane 2: Jurkat cell lysate (negative)

Lysates/proteins at 20 µg/Lane.

Predicted band size: 29 kDa
Observed band size: 50 kDa

Exposure time: 6 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 (HA723183) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Relative expression (RE)

Immunocytochemistry analysis of HUT 102 (positive) and Jurkat (negative) labeling CD134 with Rabbit anti-CD134 antibody (HA723183) at 1/500 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-CD134 antibody (HA723183) at 1/500 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.

☑ Relative expression (RE)

Flow cytometric analysis of Jurkat (left, negative) and HUT 102 (right, positive) cells labeling CD134.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA723183, 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).

CD134 was immunoprecipitated from 0.2 mg HUT 102 cell lysate with HA723183 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723183 at 1/1,000 dilution. Mouse Anti-Rabbit IgG kappa light chain secondary antibody (M1208-2) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: HUT 102 cell lysate (input)
Lane 2: HA723183 IP in HUT 102 cell lysate
Lane 3: Rabbit IgG instead of HA723183 in HUT 102 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 2 seconds; ECL: K1801