NCAM1 / CD56 Recombinant Rabbit Monoclonal Antibody [PSH06-53]

Western blot analysis of NCAM1 / CD56 on different lysates with Rabbit anti-NCAM1 / CD56 antibody (HA722756) at 1/20,000 dilution.

Lane 1: SH-SY5Y (Human neuroblastoma cell) cell lysate
Lane 2: HeLa (Human cervical adenocarcinoma cell) cell lysate
Lane 3: C6 (Rat glioma cell) cell lysate
Lane 4: Rat brain tissue lysate

Lysates/proteins at 15 µg/Lane.
Exposure time: 6 seconds; ECL: K1801

Negative expression of NCAM1 / CD56 protein in HeLa is consistent with the predicted expression pattern.

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: HA722756, 1/20,000 in primary antibody dilution buffer (K1803), overnight at 4 ℃
Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

Predicted band size: 95 kDa
Observed band size: 100-250 kDa

Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-NCAM1 / CD56 antibody (HA722756) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722756) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-NCAM1 / CD56 antibody (HA722756) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722756) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunocytochemistry analysis of SH-SY5Y cells labeling NCAM1 / CD56 with Rabbit anti-NCAM1 / CD56 antibody (HA722756) at 1/1,000 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-NCAM1 / CD56 antibody (HA722756) at 1/1,000 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 C6 cells labeling NCAM1 / CD56 with Rabbit anti-NCAM1 / CD56 antibody (HA722756) 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-NCAM1 / CD56 antibody (HA722756) 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.

☑ Relative expression (RE)

Flow cytometric analysis of HeLa (left, negative) and SH-SY5Y (right, positive) cells labeling NCAM1 / CD56.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA722756, 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™ 594 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1122) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Flow cytometric analysis of human peripheral blood lymphocytes labeling NCAM1 / CD56.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA722756, 1μg/mL) (red). 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).

NCAM1 / CD56 was immunoprecipitated from 0.2 mg SH-SY5Y cell lysate with HA722756 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722756 at 1/1,000 dilution. Anti-Rabbit IgG for IP, AlpSdAbs® VHH(HRP) (025-100-005) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: SH-SY5Y cell lysate (input)
Lane 2: HA722756 IP in SH-SY5Y cell lysate
Lane 3: Rabbit IgG instead of HA722756 in SH-SY5Y cell lysate

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