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Western blotAntibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [2]
- Immunocytochemistry [2]
- Immunohistochemistry [1]
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- Product number
- PA1-10015 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- NEFM Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant full-length protein
- Description
- In Western blot, this antibody detects bands at ~145 kDa in crude homogenates of rat cerebellum homogenate. Human and bovine run slower at ~160 kDa. A minor band at1~110 kDa is also frequently seen, which appears to be an in-vivo proteolytic fragment of NF-M.
- Reactivity
- Human, Mouse, Rat, Bovine, Chicken/Avian, Porcine
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- Conc. Not Determined
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of NEFM in neuronal tissue lysates using an NEFM polyclonal antibody (Product # PA1-10015) at a dilution of 1:2,000 as seen in green. 1) protein standard (red), 2) rat brain, 3) rat spinal cord, 4) mouse brain, 5) mouse spinal cord, 6) pig brain, 7) pig spinal cord. Strong bands at 145 kDa correspond to rodent NEFM molecules, while the NEFM of larger mammals runs at about 160 kDa.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-NEFM Monoclonal Antibody (NF-09) (Product # PA1-10015) and a 160 kDa bands corresponding to NEFM was observed in HEK-293 cell line, Mouse Cerebellum, Mouse Brain and Rat Brain but not in HeLa, Hep G2, Mouse Heart and Mouse Testis. Membrane extracts (30 µg lysate) of HEK-293 (Lane 1), HeLa (Lane 2) and Hep G2 (Lane 3), 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 (1:1000 dilution) and detected by chemiluminescence Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036), 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)
- Main image
- Experimental details
- Knockout of NEFM was achieved by CRISPR-Cas9 genome editing. Immunofluorescence analysis was performed on wild type HEK-293 cells (panel a,d), HEK-293 Cas9 cells (panels b,e) and HEK-293 NEFM KO cells (panel c,f). Cells were fixed, permeabilized, and labelled with NEFM Polyclonal Antibody (Product # PA1-10015) at 1:1000 dilution, followed by Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at 1:2000 dilution. Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415) at a dilution of 1:300 was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,f) upon CRISPR mediated knockout (KO) confirms that antibody is specific to NEFM (green). The images were captured at 60X magnification.
- 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 Rabbit Polyclonal Antibody (Product # PA1-10015) at 1:1000 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A27034) 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 Rabbit Polyclonal Antibody (Product # PA1-10015) at 1:1000 dilution. Panel g represents the merged image of HeLa cells showing no expression for NF-M protein. The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of NEFM in rat cerebellum section. The rat cerebellum section was obtained following transcardial perfusion of the rat with 4% paraformaldehyde, brain was post fixed for 24 hours, and cut to 45µM. Free-floating sections were stained using an NEFM polyclonal antibody (Product # PA1-10015) at a dilution of 1:2,000 as seen in red, and costained using a GAP43 monoclonal antibody at a dilution of 1:2,000 as seen in green. The NEFM antibody strongly labels neuronal processes throughout the cerebellum, while the GAP43 antibody stains predominantly synaptic regions in the molecular layer.
