OMA1-06110

antibody from Invitrogen Antibodies

Targeting: NEFM NEF3, NF-M, NFM

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Recommended by provider)

Antibody Data

Product number

OMA1-06110

Provider

Invitrogen Antibodies

Product name

NEFM Monoclonal Antibody (RNF 403)

Antibody type

Monoclonal

Antigen

Other

Description

OMA1-06110 detects phosphorylated 160 kDa neurofilament from human, hamster, non-human primate and xenopus tissues. OMA1-06110 has been successfully used in Western blot and immunohistochemistry (frozen) procedures. By Western blot, this antibody detects an ~160 kDa protein representing the phosphorylated form of the 160 kDa neurofilament from human nervous tissue extract. OMA1-06110 antigen is cytoskeletal preparation of calf brain tissue.

Reactivity

Human, Rat, Bovine, Hamster, Xenopus

Host

Mouse

Isotype

IgG

Antibody clone number

RNF 403

Vial size

100 µL

Concentration

1 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

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 (RNF 403) (Product # OMA1-06110) (1:500 dilution) 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)

Main image

Experimental details

Western blot was performed using Anti-NEFM Monoclonal Antibody (RNF 403) (Product # OMA1-06110) and a160 kDa band corresponding to NEFM was observed in HEK-293 cell line but not in HeLa and Hep G2 cell line which are reported to be negative. Membrane enriched extracts (30 µg lysate) of HEK-293 (Lane 1), HeLa (Lane 2) and Hep G2 (Lane 3) 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 (1:500 dilution) 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).

Immunocytochemistry

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 # OMA1-06110) 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 conjugate (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 of HEK-293 showing cytoskeletal (intermediate filaments) localization. Panel e represents the merged image of HeLa cells showing no expression for NF-M protein. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.