- MA1-19225 - Invitrogen Antibodies NEFM antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NEFM using a monoclonal antibody (Product # MA1-19225).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blotting analysis of human neurofilament M protein using mouse monoclonal antibody NF-09 on lysates of HEK-293 cell line, and MCF-7 cell line (neurofilament non-expressing cell line; negative control) under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2µg/mL of mouse anti-neurofilament M Monoclonal antibody (Product # MA1-19225) followed by IRDye800-conjugated anti-mouse secondary antibody. Neurofilament M was detected around 160 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NEFM was performed by loading 20 µg of HEK-293 wild type (Lane 1), HEK-293 Cas9 control (Lane 2), HEK-293 NEFM knockout (Lane 3) membrane enriched cell extracts. The blot was probed with Anti-NEFM Monoclonal Antibody (NF-09) (Product # MA1-19225) (1ug/mL) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177) (1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to NEFM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-NEFM Monoclonal Antibody (NF-09) (Product # MA1-19225) and a 160 kDa band corresponding to NEFM was observed in HEK-293 cell line, Mouse Cerebellum but not in HeLa, Hep G2 cell line, Mouse Heart, Mouse Testis. Figure (a) Membrane extracts (30 µg lysate) of HEK-293 (Lane 1), HeLa (Lane 2) and Hep G2 (Lane 3); Figure (b) Tissue extracts (30 µg lysate) of Mouse Cerebellum (Lane 4), Mouse Brain (Lane 5), Rat Brain (Lane 6), Mouse Heart (Lane 7), Mouse Testis (Lane 8) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). 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 (1 µg/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP(Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry staining of neurofilament medium protein in murine Neuro2A cells by NF-09 Monoclonal antibody (Product # MA1-19225) conjugated with Dyomics 547 (red). DNA stained by Hoechst (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of NF-M was performed using 70% confluent log phase HEK-293 and HeLa 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. HEK-293 cells were labeled with NF-M Mouse Monoclonal Antibody (Product # MA1-19225) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A28175) at a dilution of 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). Panel d represents the merged image showing cytoskeletal localization. Panel e represents control cells with no primary antibody to assess background. Panel f represents HeLa cells labeled with NF-M Mouse Monoclonal Antibody (Product # MA1-19225) at 5 µg/mL. Panel g represents the merged image of HeLa cells showing no expression for NF-M protein. The images were captured at 60X magnification.

MA1-19225