Immunocytochemical staining of NF-M immunoreactivity (GREEN) and Glial Fibrillary Acidic Protein (GFAP, RED) in a culture of mouse cortical neurons. The chicken anti-NFM immunostaining was performed using a 1:2000 dilution, and the GFAP immunostaining was performed using a 1:5000 dilution. Photomicrographs from Dr. Gerry Shaw, EnCor Biotechnology, Inc.
NFM protein was purified from bovine spinal cords following the method of Delacourte et al. ["Study of the 10-nm-filament fraction isolated during the standard microtubule prepartion" (1980). Biochem. J. 191(2): 543-546], followed by Prepcell purification (Bio-Rad). Antibodies were prepared by injecting purified NFM protein into laying hens and purifying the IgY fraction from eggs collected from hyper-immunized hens. After several boosts, hens were co-boosted with recombinant NFM protein expressed in bacteria. These antibodies have been validated with human, mouse and rat tissues.
Volume: 200 µL
Concentration: 2 mg/mL
Host Species: Chicken
Species Reactivity: Human, Mouse, Rat
Applications: ICC, IHC, WB
Protein Name / Synonyms: Neurofilament medium polypeptide (NF-M) (160 kDa neurofilament protein) (Neurofilament 3) (Neurofilament triplet M protein)
Target Description: Human Neurofilament Medium Chain (NFM) is a 102,448 dalton protein (916 amino acids) expressed in CNS and PNS neurons. This cytoskeletal protein is normally phosphorylated in vivo and its extent of phosphorylation correlates with different states of axonal growth and stabilization. These observations have led to the hypothesis that the NFM protein cross-links adjacent neurofilament strands and contributes to the structural integrity of the axon.
Gene ID: NEFM NEF3 NFM
Antibody Registry ID (RRID): AB_2313554
Physical State: Liquid
Production Notes: NFM protein was purified from bovine spinal cords following the method of Delacourte et al. ["Study of the 10-nm-filament fraction isolated during the standard microtubule prepartion" (1980). Biochem. J. 191(2): 543-546], followed by Prepcell purification (Bio-Rad). Antibodies were prepared by injecting purified NFM protein into laying hens. After repeated injections into the hens, immune eggs were collected, and the IgY fractions were purified from the yolks. Antibodies were diluted in the buffer described above, and then filter-sterilized.
Validation and Application Notes
Molecular Weight: 150 kDa
Western Blot Dilution Range: 1:1000-1:2000
IHC Dilution Range: 1:1000-1:2000
Quality assurance analysis was performed using immunohistochemistry (at a dilution of 1:5000) using fluorescein-labeled goat anti-chicken IgY (1:500 dilution, Aves Labs Cat.# F-1005) as the secondary reagent.
Store at 4°C in the dark. Under these conditions, the antibodies should have a shelf life of at least 12 months (provided they remain sterile). Do not freeze these antibodies unless you want to store them for longer periods of time. Note, however, that each time an antibody preparation is frozen, about half of its binding activity is lost.
Aves Labs products are intended for use as research laboratory reagents. They are not intended for use as diagnostic or therapeutic reagents in humans.
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