CD166 Mouse Monoclonal Antibody [5-5]

Western blot analysis of CD166 on different lysates with Rabbit anti-CD166 antibody (EM1902-36) at 1/5,000 dilution.

Lane 1: A549 (Human lung adenocarcinoma cell) cell lysate
Lane 2: SH-SY5Y (Human neuroblastoma cell) cell lysate
Lane 3: A431 (Human epidermoid carcinoma skin squamous cell) cell lysate
Lane 4: HaCaT (Human immortalized keratinocyte) cell lysate

Lysates/proteins at 15 µg/Lane.
Exposure time: 135 seconds; ECL: K1801

Blocking: 5% NFDM/TBST, 1 hour at room temperature
Primary antibody: EM1902-36, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 ℃
Secondary antibody: Goat anti-Mouse IgG-HRP (HA1006), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

Predicted band size: 65.1 kDa
Observed band size: 100 kDa

☑ Knockdown (KD)

Western blot analysis of CD166 on different lysates with Mouse anti-CD166 antibody (EM1902-36) at 1/1,000 dilution.

Lane 1: A549-WT cell lysate
Lane 2: A549-KD CD166 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 65 kDa
Observed band size: 100 kDa

Exposure time: 2 minutes 10 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 (EM1902-36) at 1/1,000 dilution was used in 5% BSA at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

ICC staining of CD166 in A431 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (EM1902-36, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).

ICC staining of CD166 in Huvec cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (EM1902-36, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).

Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-CD166 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (EM1902-36, 1/100) for 30 minutes 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.

Flow cytometric analysis of CD166 was done on THP-1 cells. The cells were fixed, permeabilized and stained with the primary antibody (EM1902-36, 1/100) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated goat anti-Mouse IgG Secondary antibody at 1/500 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).