Revision 1

#35574Store at -20C

1 Kit

(8 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
NeuN (D4G4O) XP® Rabbit mAb 24307 20 µl 46-55 kDa Rabbit IgG
GAP43 (D9C8) Rabbit mAb 8945 20 µl 38, 43 kDa Rabbit IgG
MAP2 (D5G1) XP® Rabbit mAb 8707 20 µl 75, 82, 280 kDa Rabbit IgG
Neurofilament-L (C28E10) Rabbit mAb 2837 20 µl 70 kDa Rabbit IgG
β3-Tubulin (D71G9) XP® Rabbit mAb 5568 20 µl 55 kDa Rabbit IgG
Synaptophysin (D8F6H) XP® Rabbit mAb 36406 20 µl 38 kDa Rabbit IgG
PSD95 (D27E11) XP® Rabbit mAb 3450 20 µl 95 kDa Rabbit IgG
UCHL1 (D3T2E) XP® Rabbit mAb 13179 20 µl 27 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Mature Neuron Marker Antibody Sampler Kit provides an economical means for detecting mature neuron proteins by western and labeling mature neuronal structures by immunofluorescence (IF). This kit includes enough primary antibodies to perform two western blot experiments or at least forty IF tests per primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibodies.

Background

The antibodies in this kit serve to characterize and identify mature neurons. Neural stem cells differentiate into mature post-mitotic neurons that are incapable of cellular division. Several neuron-enriched markers can be used to identify mature neurons. Neuronal nuclei (NeuN, Fox-3, RBFOX3) is a nuclear protein expressed in most post-mitotic neurons of the central and peripheral nervous systems. NeuN is not detected in Purkinje cells, sympathetic ganglion cells, Cajal-Retzius cells, INL retinal cells, inferior olivary, or dentate nucleus neurons (1). This neuronal protein was originally identified by immunoreactivity with a monoclonal antibody also called NeuN. Using MS-analysis, NeuN was later identified as the Fox-3 gene product, which contains an RNA recognition motif and functions as a splicing regulator (2). As neurons mature, they develop elaborate processes like axons and dendrites that are necessary to drive core neuronal functions, including synaptic transmission.

GAP43 is a nervous system specific, growth-associated protein enriched in growth cones and areas of high plasticity (3). GAP43 is integral to growth cone formation, neurite outgrowth, and the development of a functional cerebral cortex (4). The cytoskeleton, which is important in generating neuronal processes, consists of three types of cytosolic fibers: actin microfilaments, intermediate filaments, and microtubules. β3-tubulin is one of six β-tubulin isoforms that make up the building blocks of microtubules (5). Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that regulates the structure and stability of microtubules, neuronal morphogenesis, cytoskeleton dynamics, and organelle trafficking in axons and dendrites (6). MAP2 is preferentially localized to dendrites in cultured neurons (7). Neurofilaments are the major intermediate filaments found in neurons and consist of light (NFL), medium (NFM), and heavy (NFH) subunits (8). Similar in structure to other intermediate filament proteins, neurofilaments have a globular amino-terminal head, a central α-helical rod domain, and a carboxy-terminal tail. A heterotetrameric unit (NFL-NFM and NFL-NFH) forms a protofilament, with eight protofilaments comprising the typical 10 nm intermediate filament (9). Neurofilaments are critical for radial axon growth and determine axon caliber, serving as markers for neuronal axons.

Mature neurons function as cellular mediators of synaptic transmission. Synaptophysin is a neuronal synaptic vesicle glycoprotein (10). Synaptophysin is responsible for targeting synaptobrevin 2/VAMP2 to synaptic vesicles, and is a critical component and marker for the presynaptic fusion complex (11). Postsynaptic Density protein 95 (PSD95) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. These family members consist of an amino-terminal variable segment followed by three PDZ domains, an SH3 domain, and an inactive guanylate kinase (GK) domain. PSD95 is a scaffolding protein involved in the assembly and function of mature postsynaptic density complexes (12,13).

Several cellular processes are required to support dynamic functions existing in mature neurons, including protein regulation by protein ubiquitination. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function (14).

  1. Mullen, R.J. et al. (1992) Development 116, 201-11.
  2. Kim, K.K. et al. (2009) J Biol Chem 284, 31052-61.
  3. Biewenga, J.E. et al. (1996) Acta Biochim Pol 43, 327-38.
  4. Aigner, L. and Caroni, P. (1993) J Cell Biol 123, 417-29.
  5. Jiang, Y.Q. and Oblinger, M.M. (1992) J Cell Sci 103 (Pt 3), 643-51.
  6. Sánchez, C. et al. (2000) Prog Neurobiol 61, 133-68.
  7. Caceres, A. et al. (1984) Brain Res 315, 314-8.
  8. Al-Chalabi, A. and Miller, C.C. (2003) Bioessays 25, 346-55.
  9. Cohlberg, J.A. et al. (1995) J Biol Chem 270, 9334-9.
  10. Wiedenmann, B. and Franke, W.W. (1985) Cell 41, 1017-28.
  11. Bonanomi, D. et al. (2007) Biochem J 404, 525-34.
  12. Cao, J. et al. (2005) J Cell Biol 168, 117-26.
  13. Chetkovich, D.M. et al. (2002) J Neurosci 22, 6415-25.
  14. Gong, B. et al. (2006) Cell 126, 775-88.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #35574

    Mature Neuron Marker Antibody Sampler Kit

    Mature Neuron Marker Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from mouse brain using PSD95 (D27E11) XP® Rabbit mAb #3450. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
    Mature Neuron Marker Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from mouse brain using Synaptophysin (D8F6H) XP® Rabbit mAb #36406. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    Mature Neuron Marker Antibody Sampler Kit: Image 3 Expand Image
    Immunoprecipitation of GAP43 from SH-SY-5Y extracts. Lane 1 is #8945 GAP43 (D9C8) Rabbit mAb, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is 10% input. Western blot analysis was performed with GAP43 (DA1E) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.
    Mature Neuron Marker Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using UCHL1 (D3T2E) XP® Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Mature Neuron Marker Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from mouse brain, C2C12 cells, and rat brain using NeuN (D4G4O) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mature Neuron Marker Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from mouse brain, HeLa cells and rat brain, using Neurofilament-L (C28E10) Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from human cerebellum and rat brain using PSD95 (D27E11) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from mouse and rat tissue using Synaptophysin (D8F6H) XP® Rabbit mAb (upper) and B-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mature Neuron Marker Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of HeLa and human cerebellum using β3-Tubulin (D71G9) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 10 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Mature Neuron Marker Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from neonatal and adult mouse brain using MAP2 (D5G1) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from mouse neonatal brain and rat fetal brain using GAP43 (D9C8) Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human UCHL1 (hUCHL1-Myc/DDK; +), Myc/DDK-tagged full-length human UCHL3 (hUCHL3-Myc/DDK; +), Myc/DDK-tagged full-length human UCHL5 (hUCHL5-Myc/DDK; +), and Myc/DDK-tagged full-length human BAP1 (hBAP1-Myc/DDK; +), using UCHL1 (D3T2E) XP® Rabbit mAb (upper) or DYKDDDDK Tag Antibody #2368 (lower).
    Mature Neuron Marker Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse cerebellum using NeuN (D4G4O) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded cynomolgus monkey brain using NeuN (D4G4O) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain using Neurofilament-L (C28E10) Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 17 Expand Image
    Confocal immunofluorescent analysis of rat cerebellum and retina using PSD95 (D27E11) XP® Rabbit mAb (red) and Neurofilament-L (DA2) Mouse mAb #2835 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded Neuro-2a cell pellet (left, positive) or C2C12 cell pellet (right, negative) using Synaptophysin (D8F6H) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescent analysis of mouse cerebellum using β3-Tubulin (D71G9) XP® Rabbit mAb (green) and Tau (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of frozen rat cerebellum using MAP2 (D5G1) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 21 Expand Image
    Confocal immunofluorescent analysis of rat hippocampus (left) and retina (right) using GAP43 (D9C8) Rabbit mAb (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 555 Conjugate) #3656 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from Ramos and K-562 cells using UCHL1 (D3T2E) XP® Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). As expected, UCHL1 protein expression is not detected in K-562 cells.
    Mature Neuron Marker Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon (myenteric plexus) using NeuN (D4G4O) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human brain using Neurofilament-L (C28E10) Rabbit mAb in the presence of control peptide (left) or Neurofilament-L blocking peptide #1005 (right).
    Mature Neuron Marker Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse cerebellum using Synaptophysin (D8F6H) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 26 Expand Image
    Confocal immunofluorescent analysis of P19 cells that were differentiated with retinoic acid, using β3-Tubulin (D71G9) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5 #4084® (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using UCHL1 (D3T2E) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human glioblastoma multiform using NeuN (D4G4O) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 29 Expand Image
    Confocal immunofluorescent analysis of normal rat cerebellum using Neurofilament-L (C28E10) Rabbit mAb (green) and GFAP (GA5) Mouse mAb #3670 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon using Synaptophysin (D8F6H) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded DU 145 (left; positive) and LNCaP (right; negative) cell pellets using UCHL1 (D3T2E) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 32 Expand Image
    Confocal immunofluorescent analysis of mouse hippocampus (left), cortex (middle), and cerebellum (right) using NeuN (D4G4O) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 33 Expand Image
    Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using Synaptophysin (D8F6H) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 34 Expand Image
    Immunoprecipitation of β3-Tubulin from mouse brain tissue extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is β3-Tubulin (D71G9) XP® Rabbit mAb. Western blot analysis was performed using β3-Tubulin (D71G9) XP® Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as the secondary antibody.
    Mature Neuron Marker Antibody Sampler Kit: Image 35 Expand Image
    Immunohistochemical analysis of paraffin-embedded colorectal adenocarcinoma using UCHL1 (D3T2E) XP® Rabbit mAb in the presence of control peptide (left) or antigen specific peptide (right).
    Mature Neuron Marker Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using Synaptophysin (D8F6H) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 37 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate adenocarcinoma using UCHL1 (D3T2E) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 38 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse pancreas using Synaptophysin (D8F6H) XP® Rabbit mAb.
    Mature Neuron Marker Antibody Sampler Kit: Image 39 Expand Image
    Confocal immunofluorescent analysis of mouse brain using UCHL1 (D3T2E) XP® Rabbit mAb (green). Blue = Hoescht 33342 #4082 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 40 Expand Image
    Confocal immunofluorescent analysis of mouse retina using Synaptophysin (D8F6H) XP® Rabbit mAb (green), Neurofilament-L (DA2) Mouse mAb #2835 (yellow) and Lamin A/C (4C11) Mouse mAb #4777 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Mature Neuron Marker Antibody Sampler Kit: Image 41 Expand Image
    Confocal immunofluorescent analysis of mouse brain using UCHL1 (D8R2I) XP® Rabbit mAb (green). Blue = Hoescht 33342 #4082 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 42 Expand Image
    Confocal immunofluorescent analysis of mouse brain using Synaptophysin (D8F6H) XP® Rabbit mAb (green), Neurofilament-L (DA2) Mouse mAb #2835 (yellow) and Lamin A/C (4C11) Mouse #4777 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Mature Neuron Marker Antibody Sampler Kit: Image 43 Expand Image
    Confocal immunofluorescent analysis of mouse olfactory bulb (left) and pons (right) using UCHL1 (D3T2E) XP® Rabbit mAb #13179 (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 555 Conjugate) #3656 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Mature Neuron Marker Antibody Sampler Kit: Image 44 Expand Image
    Confocal immunofluorescent analysis of DU 145 (positive; left) and LNCaP (negative; right) cells using UCHL1 (D3T2E) XP® Rabbit mAb (green). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
    Mature Neuron Marker Antibody Sampler Kit: Image 45 Expand Image
    Flow cytometric analysis of K-562 cells (blue; negative) and Ramos cells (green; positive) using UCHL1 (D3T2E) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.