Other Immunogen Type corresponding to Pig Hypophosphorylated Neurofilament H (C terminal). C-terminal segment of enzymatically dephosphorylated pig Neurofilament 200.
Other applications have not been tested.
Optimal dilutions should be determined by end users.
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Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 0.5 µg/ml. Detects a band of approximately 200 kDa (predicted molecular weight: 112 kDa).
Use a concentration of 1 - 2 µg/ml.
Use a concentration of 1 - 2 µg/ml. Acetone fixed.
Use 1µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration.
Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber. NF-H has an important function in mature axons that is not subserved by the two smaller NF proteins.
Defects in NEFH are a cause of susceptibility to amyotrophic lateral sclerosis (ALS) [MIM:105400]. ALS is a neurodegenerative disorder affecting upper and lower motor neurons, and resulting in fatal paralysis. Sensory abnormalities are absent. Death usually occurs within 2 to 5 years. The etiology is likely to be multifactorial, involving both genetic and environmental factors.
Belongs to the intermediate filament family.
There are a number of repeats of the tripeptide K-S-P, NFH is phosphorylated on a number of the serines in this motif. It is thought that phosphorylation of NFH results in the formation of interfilament cross bridges that are important in the maintenance of axonal caliber. Phosphorylation seems to play a major role in the functioning of the larger neurofilament polypeptides (NF-M and NF-H), the levels of phosphorylation being altered developmentally and coincident with a change in the neurofilament function. Phosphorylated in the Head and Rod regions by the PKC kinase PKN1, leading to inhibit polymerization.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Hypophosphorylated Neurofilament H antibody [N52] (ab82259)This image is courtesy of an anonymous Abreview
ab82259 staining Hypophosphorylated Neurofilament H in Rat nerve tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, permeablized with 0.2 Triton-X and blocked with 10% serum. The sample was incubated with primary antibody (1/50 in PBS plus 1x Casien) at 25°C for 2 hours. An Alexa Fluor®488-conjugated Goat anti-mouse IgG polyclonal(1/200) was used as the secondary antibody. GFAP was labelled with Texas Red.
Immunocytochemistry/ Immunofluorescence - Anti-Hypophosphorylated Neurofilament H antibody [N52] (ab82259)
ICC/IF image of ab82259 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab82259, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Flow Cytometry - Anti-Hypophosphorylated Neurofilament H antibody [N52] (ab82259)
Overlay histogram showing SH-SY5Y cells stained with ab82259 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab82259, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
Prest TA et al. Nerve-specific, xenogeneic extracellular matrix hydrogel promotes recovery following peripheral nerve injury. J Biomed Mater Res A106:450-459 (2018).
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