Neurofilament-M/H Antibody [G10L12]

Application: Reactivity: Human, Mouse, Rat

Usage Information

Dilution
WB1:1000 IF1:100

Application
WB, IHC, IF

Reactivity
Human, Mouse, Rat

Source
Mouse Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
102 kDa 160 kDa, 200 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Datasheet & SDS

Biological Description

Specificity
Neurofilament-M/H Antibody [G10L12] detects endogenous levels of total Neurofilament-M/H protein.

Clone
G10L12

Synonym(s)
Neurofilament medium polypeptide, NF-M, 160 kDa neurofilament protein, Neurofilament 3, Neurofilament triplet M protein, NEFM, NEF3, NFM

Background
Neurofilament-M and neurofilament-H are neuron-specific type IV intermediate filament subunits that assemble with neurofilament-L and α‑internexin into 10‑nm heteropolymers forming the major cytoskeletal system of large myelinated axons, where they regulate axonal caliber and contribute to the mechanical stability and conduction properties of long projection neurons. Each subunit contains a conserved central α‑helical rod domain that drives coiled-coil dimer formation and filament assembly, flanked by an N‑terminal head and an extended C‑terminal tail; NF‑M and NF‑H are distinguished by long, intrinsically disordered tail domains that project laterally from the filament core and are densely populated with Lys–Ser–Pro (KSP) repeat motifs that serve as major sites for multi-kinase phosphorylation. The KSP-rich tails of NF‑M and NF‑H are phosphorylated by several kinases, including PKA, PKC, Cdk5, ERK, GSK‑3, SAPK, and PKN, and this post-translational modification modulates interfilament spacing, interaction with microtubules and actin, and resistance to proteolysis, thereby tuning the packing density of neurofilaments within axons and stabilizing the stationary neurofilament network along mature axons. Deletion or truncation of the C‑terminal tail domains of both NF‑H and NF‑M reduces steady-state neurofilament content along optic axons without altering subunit synthesis or slow axonal transport rates and accelerates clearance of neurofilaments from the stationary cytoskeleton, indicating that these tails are required to maintain neurofilament levels by blocking turnover rather than by regulating transport. NF‑M and NF‑H thereby support radial growth and caliber maintenance of myelinated axons and influence conduction velocity by determining the number and spacing of neurofilaments and their cross-bridging interactions, while also affecting the dynamics and function of other cytoskeletal elements such as microtubules through physical association and shared regulatory kinases. Neurofilament subunits, including NF‑M and NF‑H, are expressed primarily in neurons and display region- and caliber-dependent abundance, and their phosphorylation state exhibits proximal–distal gradients along axons that correlate with maturation, myelination, and segment-specific mechanical requirements. Perturbations in NF‑M/H expression, tail phosphorylation, or turnover are frequent in neurodegenerative conditions, where downregulation of neurofilament mRNAs and accumulation of disorganized or aggregated neurofilaments are described in amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease, and interference with axonal transport by disorganized neurofilament bundles is proposed as one mechanism contributing to selective axonal and neuronal loss. Elevated levels of neurofilament proteins and their phosphorylated fragments in cerebrospinal fluid and blood serve as biomarkers of axonal damage and disease progression across a range of motor neuron and neurodegenerative disorders.

Background

Neurofilament-M and neurofilament-H are neuron-specific type IV intermediate filament subunits that assemble with neurofilament-L and α‑internexin into 10‑nm heteropolymers forming the major cytoskeletal system of large myelinated axons, where they regulate axonal caliber and contribute to the mechanical stability and conduction properties of long projection neurons. Each subunit contains a conserved central α‑helical rod domain that drives coiled-coil dimer formation and filament assembly, flanked by an N‑terminal head and an extended C‑terminal tail; NF‑M and NF‑H are distinguished by long, intrinsically disordered tail domains that project laterally from the filament core and are densely populated with Lys–Ser–Pro (KSP) repeat motifs that serve as major sites for multi-kinase phosphorylation. The KSP-rich tails of NF‑M and NF‑H are phosphorylated by several kinases, including PKA, PKC, Cdk5, ERK, GSK‑3, SAPK, and PKN, and this post-translational modification modulates interfilament spacing, interaction with microtubules and actin, and resistance to proteolysis, thereby tuning the packing density of neurofilaments within axons and stabilizing the stationary neurofilament network along mature axons. Deletion or truncation of the C‑terminal tail domains of both NF‑H and NF‑M reduces steady-state neurofilament content along optic axons without altering subunit synthesis or slow axonal transport rates and accelerates clearance of neurofilaments from the stationary cytoskeleton, indicating that these tails are required to maintain neurofilament levels by blocking turnover rather than by regulating transport. NF‑M and NF‑H thereby support radial growth and caliber maintenance of myelinated axons and influence conduction velocity by determining the number and spacing of neurofilaments and their cross-bridging interactions, while also affecting the dynamics and function of other cytoskeletal elements such as microtubules through physical association and shared regulatory kinases. Neurofilament subunits, including NF‑M and NF‑H, are expressed primarily in neurons and display region- and caliber-dependent abundance, and their phosphorylation state exhibits proximal–distal gradients along axons that correlate with maturation, myelination, and segment-specific mechanical requirements. Perturbations in NF‑M/H expression, tail phosphorylation, or turnover are frequent in neurodegenerative conditions, where downregulation of neurofilament mRNAs and accumulation of disorganized or aggregated neurofilaments are described in amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease, and interference with axonal transport by disorganized neurofilament bundles is proposed as one mechanism contributing to selective axonal and neuronal loss. Elevated levels of neurofilament proteins and their phosphorylated fragments in cerebrospinal fluid and blood serve as biomarkers of axonal damage and disease progression across a range of motor neuron and neurodegenerative disorders.

References
https://pubmed.ncbi.nlm.nih.gov/28373358/ https://pubmed.ncbi.nlm.nih.gov/23028520/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%