EnCorBio/Mouse Monoclonal Antibody to FOX3/NeuN Cat# MCA-1B7/50µL of 1mg/mL/MCA-1B7188bio精品生物—专注于实验室精品爆款的电商平台

Mouse Monoclonal Antibody to FOX3/NeuN Cat# MCA-1B7

$120.00 – $800.00

In the early 90s an unusual protocol resulted in the raising of a mouse monoclonal antibody, clone A60, against a component of neuronal nuclei and proximal perikarya (1). The component was therefore named “NeuN” and was shown to correspond to two protein bands at 46 and 48kDa on western blots. The antibody become very widely used as a reliable neuronal marker, apparently binding to neurons in all vertebrates, and athough a few neuronal cell types were not recognized such as cerebellar Purkinje cells, olfactory mitral cells and many type of retinal neuron, NeuN immunoreactivity has been widely used to identify neurons. The identity of the NeuN protein was unknown until 2009 when Kim et al. (2) showed that it was identical to FOX3, a mammalian homolog of a gene product originally identified in C. elegans and named FOX1 (2,3). There are three mammalian homologs, FOX1, FOX2 and FOX3, which are believed to have a role in the regulation of mRNA splicing (4). All three contain an almost identical central RNA recognition motif or RRM domain, a region of about 90 amino acids found in numerous proteins which in all three molecules specifically binds the hexaribonucleotide UGCAUG (4). Four protein isoforms of FOX3 result from alternate splicing of two exons from the single gene which code for an insert close to the C-terminus and a short C-terminal extension (5). The extension includes a C-terminal proline-tyrosine sequence preceded by hydrophobic amino acids (Φ-PY) which is known to function in nuclear localization, apparently accounting for FOX3 being present in both nuclei and cytoplasm in certain neurons.The MCA-1B7 antibody was raised against a recombinant human FOX3/NeuN construct based only on the N-terminal sequence, not including the RRM domain and C-terminal regions. The N-terminal regions of FOX1, FOX2 and FOX3 are relatively poorly conserved so we were able to obtain antibodies which recognized FOX3 but not FOX2 or FOX1. The peptide YPPAQYPPPPQNGIPAEYAP, amino acids 5-24, inhibits binding of both MCA-1B7 and the original NeuN antibody to recombinant human FOX3. The central 10 amino acids of the peptide is likely the most significant component of the MCA-1B7 epitope (see here for details). The equilibrium dissociation constant (KD) is 2.69 x 10^-9M for MCA-1B7, showing significantly higher affinity than the original Millipore NeuN antibody, which has a KD of 7.95 x 10^-9M under identical conditions. We used the same recombinant immunogen to generate rabbit and chicken polyclonal antibodies to FOX3/NeuN, RPCA-FOX3, and CPCA-FOX3 respectively. These two antibodies also work in the same way as MCA-1B7 and the original NeuN antibody and are versatile reagents which can be used in double and triple staining protocols and also work well on mouse tissues on which mouse monoclonals present technical problems. Any of our FOX3/NeuN reagents can be been used to quantify the neuron/glial ratio in the brains of rats, humans and other species (6,7). Mouse select image at left for larger view.

SKU: mca-1b7Categories: Cell Type Marker, Developmental Marker, Epitope Mapped Antibodies, Kinetics, Mouse Monoclonal AntibodiesTag: Active

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Additional Info
References
Data Sheets

Name:	Mouse monoclonal antibodyto FOX3/NeuN
Immunogen:	N-terminal 99 amino acids of human FOX3 expressed in and purified from E. coli
HGNC Name:	RBFOX3
UniProt:	A6NFN3
Molecular Weight:	46, 48kDa
Host:	Mouse
Isotype:	IgG2b, κ
Species Cross-Reactivity:	Human, Rat, Mouse
RRID:	AB_2572267
Format:	Purified antibody at 1mg/mL in 50% PBS, 50% glycerol plus 5mM NaN₃
Applications:	WB, IF/ICC, IHC
Recommended Dilutions:	WB: 1:1,000 IF/ICC and IHC: 1:1,000-1:2,000
Storage:	Store at 4°C for short term, for longer term at -20°C.

FOX3 is one of a family of mammalian homologues of FOX-1, which was originally discovered in C. elegans as a gene involved in sex determination. FOX is an acronym of “Feminizing locus on X”. The mammalian genome contains three genes homologous to C. elegans FOX-1, usually called FOX1, FOX2 and FOX3. All three include a central highly conserved ~90 amino acid RRM type RNA recognition motif, identical between the three proteins, with more variability at the N and C termini. The FOX proteins are believed to have a role in the regulation of mRNA splicing. Much interest has focused on Fox3 as a result of the finding that this protein corresponds to NeuN, a neuronal nuclear antigen. The NeuN antigen was first described in 1994 by Mullen et al. (1), who raised a series of monoclonal antibodies to mouse tissues with the original intent of finding mouse species specific markers useful for transplantation experiments. In the event they obtained a clone, called mAb A60, which proved to bind an antigen expressed only in neuronal nuclei and to a lesser extent the cytoplasm of neuronal cells, and which appeared to work on all vertebrates. A few neuronal cell types were not recognized by the NeuN antibody, such as cerebellar Purkinje cells, olfactory mitral cells and retinal photoreceptors. However, the vast majority of neurons are strongly NeuN positive, and NeuN immunoreactivity has been widely used to identify neurons and measure the neuron/glial ratio in brain regions (5). The identity of the protein bound by this antibody was not characterized for many years, though the molecular weight was known to be 46 and 48kDa from western blotting experiments. Despite no knowledge about the molecular identity of NeuN, the mAb A66 antibody become very widely used as a robust marker of neurons and neuronal stem cells, and a recent Medline search using the keyword “NeuN” produced over 2900 hits.

This antibody has become widely used as sold by EnCor and through our numerous OEM partners, and information on this can be viewed using Google scholar by searching for “1B7 AND (NeuN or FOX3) or by selecting here.

Kinetic Binding DataWe used the data shown below to derive dissociation equilibrium constants (K_D values) for specified mouse monoclonal antibodies. The K_D is simply the ratio of the dissociation rate (k_off) to the association rate (k_on). Thus, K_D and affinity are inversely related so that the lower the K_D value, the higher the affinity of the antibody for its target. The figures below summarize binding data for EnCor’s mouse monoclonal antibody to FOX3/NeuN (MCA-1B7) and Millipore’s mouse monoclonal antibody to the same target (MAB377, clone A60). Experiments were performed in parallel under identical conditions. Note that EnCor’s MCA-1B7 has a significantly higher affinity for FOX3 (K_D = 3nM) versus Millipore’s MAB377 (K_D = 8nM).

Above: Binding curve set for MCA-1B7 (25nM IgG) and limiting dilutions of recombinant FOX3 peptide (0-500nM) obtained using our in-house label-free bio-layer interferometry system (Octet RED96). Color-coded traces show sensorgram data normalized to baseline after subtraction of 0nM IgG signal from all channels. Traces with overlying fit lines in red indicate their inclusion in the global fit analysis used to derive kinetic parameters listed under the legend (R^2 – goodness of correlation between the fit and data; kon – association rate constant; koff – dissociation rate constant; KD = koff/kon – affinity constant/equilibrium dissociation constant; see EnCor’s validation pipeline for more details). Mouse click on the image to get an enlarged view.

Above: Binding curve set for the Millipore mouse monoclonal antibody MAB377 (25nM IgG) and limiting dilutions of recombinant FOX3 peptide (0-500nM) obtained using our in-house label-free bio-layer interferometry system (Octet RED96). Color-coded traces show sensorgram data normalized to baseline after subtraction of 0nM IgG signal from all channels. Traces with overlying fit lines in red indicate their inclusion in the global fit analysis used to derive kinetic parameters listed under the legend (R^2 – goodness of correlation between the fit and data; kon – association rate constant; koff – dissociation rate constant; KD = koff/kon – affinity constant/equilibrium dissociation constant; see EnCor’s validation pipeline for more details). Mouse click on the image to get an enlarged view.

1. Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201-11 (1994).2. Hodgkin J, Zellan JD, Albertson DG. Identification of a candidate primary sex determination locus, fox-1, on the X chromosome of Caenorhabditis elegans. Development 120:3681-3689 (1994).3. Kim KK, Adelstein RS, Kawamoto S. Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors. J. Biol. Chem. 284:31052-61 (2009).4. Underwood JG, et al. Homologues of the Caenorhabditis elegans Fox-1 protein are neuronal splicing regulators in mammals. Mol. Cell Biol. 25:10005-16 (2005).5. Dredge BK, Jensen KB. NeuN/Rbfox3 nuclear and cytoplasmic isoforms differentially regulate alternative splicing and nonsense-mediated decay of Rbfox2. PLoS One doi: 10.1371/journal.pone.0021585 (2011).6. Herculano-Houzel S, Lent R. Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain. J. Neurosci. 25:2518-2521 (2005).7. Azevedo FA. et al. Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain.J. Comp. Neurol. 513:532-41 (2009).

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EnCorBio/Mouse Monoclonal Antibody to FOX3/NeuN Cat# MCA-1B7/50µL of 1mg/mL/MCA-1B7

Mouse Monoclonal Antibody to FOX3/NeuN Cat# MCA-1B7