CD276 Recombinant Rabbit Monoclonal Antibody [PSH02-46]

☑ Relative expression (RE)

Western blot analysis of CD276 on different lysates with Rabbit anti-CD276 antibody (HA721823) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: MCF7 cell lysate
Lane 3: HEK-293 cell lysate
Lane 4: LoVo cell lysate
Lane 5: U-2 OS cell lysate
Lane 6: LNCaP cell lysate
Lane 7: SH-SY5Y cell lysate
Lane 8: THP-1 cell lysate
Lane 9: HCT 116 cell lysate
Lane 10: Raji cell lysate (negative)

Lysates/proteins at 20 µg/Lane.

Predicted band size: 57 kDa
Observed band size: 100 kDa

Exposure time: 42 seconds; ECL: K1802;

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 (HA721823) 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.

☑ Knockdown (KD)

Western blot analysis of CD276 on different lysates with Rabbit anti-CD276 antibody (HA721823) at 1/5,000 dilution.

Lane 1: HAP1-parental cell lysate (10 µg/Lane)
Lane 2: HAP1-CD276 KD cell lysate (10 µg/Lane)

Predicted band size: 57 kDa
Observed band size: 100 kDa

Exposure time: 180 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 (HA721823) at 1/5,000 dilution was used in 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 THP-1 cells labeling CD276 with Rabbit anti-CD276 antibody (HA721823) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-CD276 antibody (HA721823) 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 (M1305-2, 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.

Immunocytochemistry analysis of MCF7 cells labeling CD276 with Rabbit anti-CD276 antibody (HA721823) at 1/100 dilution.

Cells were fixed in 100% precooled methanol for 5 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-CD276 antibody (HA721823) 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 (M1305-2, 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 THP-1 cells labeling CD276.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA721823, 1μg/mL) (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).