MA1-33723

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

MA1-33723

Provider

Invitrogen Antibodies

Product name

NEFM Monoclonal Antibody (3H11)

Antibody type

Monoclonal

Antigen

Recombinant protein fragment

Description

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222-49) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333-57) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Reactivity

Human, Mouse, Rat, Chicken/Avian

Host

Mouse

Isotype

IgG

Antibody clone number

3H11

Vial size

50 µL

Concentration

Conc. Not Determined

Storage

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

References

Submitted references

Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines.

Selvakumaran M, Pisarcik DA, Bao R, Yeung AT, Hamilton TC
Cancer research 2003 Mar 15;63(6):1311-6

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Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells.

Shaw G, Morse S, Ararat M, Graham FL
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2002 Jun;16(8):869-71

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Compartmentation of alpha-internexin and neurofilament triplet proteins in cultured hippocampal neurons.

Benson DL, Mandell JW, Shaw G, Banker G
Journal of neurocytology 1996 Mar;25(3):181-96

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot using NEFM Monoclonal Antibody (3H11) (Product # MA1-33723). Rat spinal cord homogenate showing the major intermediate filament proteins of the nervous system (lane 1). The remaining lanes show blots of this material stainted with various antibodies including NEFM Monoclonal Antibody (3H11) (Product # MA1-33723) (lane 3).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of NEFM was performed by loading 30 µg of HEK-293 wildtype control (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 (3H11)(Product # MA1-33723) using 1:2500 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 (3H11) (Product # MA1-33723) and a 160 kDa band corresponding to NEFM was observed in HEK-293 cell line, Mouse Cerebellum, Rat Brain but not in HeLa, Hep G2 cell line, Mouse Heart and Mouse Spleen. Membrane extracts (30 µg lysate) of HEK-293 (Lane 1), HeLa (Lane 2) and Hep G2 (Lane 3), Mouse Cerebellum (Lane 4), Mouse Brain (Lane 5), Rat Brain (Lane 6), Mouse Heart (Lane 7) and Mouse Spleen (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 (1:2500) 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

Immunofluorescent analysis of NEF3 in cultures of adult neural cells using a NEF3 monoclonal antibody (Product # MA1-33723). The surrounding stellate red cells are stained with rabbit polyclonal Rat facial nucleus before RPCA-a-Int. These are apparently mitotic neuronal progenitor cells and express many other neuronal markers.

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 # MA1-33723) at 1:250 dilution 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.