- MA1-17762 - Invitrogen Antibodies CEACAM6 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of CEACAM6 was achieved by transfecting A549 with CEACAM6 specific siRNAs (Silencer® select Product # s9283, s9285). Western blot analysis (Fig. a) was performed using whole cell extracts from the CEACAM6 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with CEACAM5/CEACAM6 Monoclonal Antibody (MUS) (Product # MA1-17762, 4 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Carcinoembryonic antigen-related cell adhesion molecule 6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-CEACAM5/CEACAM6 Monoclonal Antibody (MUS) (Product # MA1-17762) and a 60 kDa band corresponding to CEACAM6 was observed in A549 and MCF7 but not in MIA PaCa-2 or Hep G2 which are reported to be low expressing for CEACAM6. A uncharacterized band (*) at ~58 kDa is detected across the cell lines tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), MCF7 (Lane 2), MIA PaCa-2 (Lane 3) and Hep G2 (Lane 4) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (4 µg/mL dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with CEACAM5/CEACAM6 Monoclonal Antibody (MUS) (Product # MA1-17762) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2500), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing lysosome like punctate structures in the cytoplasm for CEACAM6 in A549. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of CEACAM6 in BOSC23 cells using a CEACAM5/CEACAM6 Monoclonal Antibody (Product # MA1-17762) at a 1.2 µg/10^6 cells dilution, detected with a PE-conjugated secondary antibody, as shown in red. Isotype control antibody is shown in black.

MA1-17762