- MA1-10050 - Invitrogen Antibodies AHNAK antibody

Lajoie JM, Cho YK, Frost D, Bremner S, Li L, Shusta EV
Protein engineering, design & selection : PEDS 2019 Dec 31;32(5):219-230

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of AHNAK was achieved by transfecting HaCaT with AHNAK specific siRNAs (Silencer® select Product # S41427, S41428). Western blot analysis (Fig. a) was performed using whole cell extracts prepared with RIPA buffer, from the AHNAK knockdown cells (lane 2) and non-targeting scrambled siRNA transfected cells (lane 1). The blot was probed with AHNAK Monoclonal Antibody (EM-09) (Product # MA1-10050, 1:2000) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:20,000) and detected by chemiluminescence using the iBright™ FL1500 Imaging System (Product # A44115). 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 AHNAK.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using AHNAK Monoclonal Antibody (EM-09) (Product # MA1-10050) and a 680 kDa band corresponding to AHNAK was observed across cell lines tested. Whole cell extracts (60 µg lysate), prepared with RIPA buffer, of U-87 MG (Lane 1), PANC-1 (Lane 2), 769-P (Lane 3), NIH:OVCAR-3 (Lane 4), HaCaT (Lane 5), SW480 (Lane 6), Jurkat (Lane 7), A-673 (Lane 8), HEL 92.1.7 (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX), 10 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:20,000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556). As reported, the levels of AHNAK were low in Jurkat, A-673 and HEL.92.17 cell lines as shown here in the figure.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence staining of AHNAK1 in human primary fibroblasts using anti-AHNAK1 (EM-09; green). Actin filaments were decorated by phalloidin (red) and cell nuclei stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence staining of AHNAK1 in HeLa cell line using anti-AHNAK1 (EM-09; red). Cell nuclei stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry staining of AHNAK1 in HeLa cell line using anti-AHNAK1 (EM-09; red) Monoclonal antibody (Product # MA1-10050). Cell nuclei stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of AHNAK was performed using 70% confluent log phase HaCaT cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with AHNAK Monoclonal Antibody (EM-09) (Product # MA1-10050, 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, 1:2000), 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 cell membrane and cytosolic localization. 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)
Main image
Experimental details: Immunocytochemistry staining of AHNAK1 in primary human fibroblasts using anti-AHNAK1 (EM-09; green) Monoclonal antibody (Product # MA1-10050). Actin filaments were decorated by phalloidin (red) and cell nuclei stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry staining (frozen sections) of murine tongue by anti-AHNAK1 antibody (EM-09; red). Actin filaments were decorated by phalloidin (green), cell nuclei stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry staining (frozen sections) of murine tongue by anti-AHNAK1 (EM-09; red) Monoclonal antibody (Product # MA1-10050). Actin filaments were decorated by phalloidin (green), cell nuclei stained with DAPI (blue).

Supportive validation

Supportive validation

MA1-10050