B7-H6 Recombinant Rabbit Monoclonal Antibody [PSH10-89]

☑ Relative expression (RE)

Western blot analysis of B7-H6 on different lysates with Rabbit anti-B7-H6 antibody (HA723256) at 1/2,000 dilution.

Lane 1: K-562 cell lysate
Lane 2: HepG2 cell lysate
Lane 3: MCF7 cell lysate (negative)
Lane 4: HeLa cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 51 kDa
Observed band size: 75 kDa

Exposure time: 2 minutes 41 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 (HA723256) 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 K-562 (positive) and Daudi (negative) labeling B7-H6 with Rabbit anti-B7-H6 antibody (HA723256) 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-B7-H6 antibody (HA723256) 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.

☑ Relative expression (RE)

Flow cytometric analysis of MCF7 (left, negative) and K-562 (right, positive) cells labeling B7-H6.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA723256, 1/5,000) (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).

B7-H6 was immunoprecipitated from 0.2 mg K-562 cell lysate with HA723256 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723256 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: K-562 cell lysate (input)
Lane 2: HA723256 IP in K-562 cell lysate
Lane 3: Rabbit IgG instead of HA723256 in K-562 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 3 minutes; ECL: K1801