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Mouse monoclonal antibody against NEUROD1: 7000PU

Mouse monoclonal antibody against NEUROD1: 7000PU

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Mouse monoclonal antibody against NEUROD1 (Neurogenic differentiation factor 1) also known as BETA2 (Beta-Cell E-Box Transactivator 2), BHF-1, MODY6, bHLHa3 is a class A basic helix loop helix transcription factor.

 

Product Information

 

Product Number:                    7000PU

 

Size:                                        100ug

 

Concentration:                       0.5 mg/ml.

 

Storage Buffer:                       Phosphate buffered saline, pH 7.2 with 0.05% sodium azide.

 

Clone:                                     BC18-1.11EG (abbreviated 1.11EG)

 

Host:                                       Mouse

Immunogen:                           Recombinant NeuroD1 protein

Isotype:                                   Mouse IgG1, Kappa

 

Reactivity:  The antibody was tested against several human cancer cell lines, cryosections of human skeletal muscle tissues, mouse cell lines and pancreatic tissue. Not tested in other species.

 

Applications: Indirect immunofluorescence, immunohistochemistry & western blotting. The antibody reacts by ELISA against recombinant protein but not tested against the authentic protein. Not tested for use in immunoprecipitation. Suggested starting concentrations are as follows; Western blotting 1 -2ug/ml, immunohistochemistry 2-5ug/ml. The 1.11EG antibody may be used on cryosections, tissue culture cells grown on chamber slides or attached on adhesion slides. Not tested on formalin fixed paraffin embedded (FFPE) tissue sections. Cultured cells and cryosections should be fixed in 1-2% paraformaldehyde for 30 mins, permeabilized in 0.25% Triton X 100 in PBS for 30 mins and non-specific antibody binding blocked with 1% BSA in PBS. The primary antibody may be used at 3-5ug/ml in 1%BSA in PBS.

 

Preparation and Storage: The monoclonal antibody was purified from tissue culture supernatant using Protein G Sepharose columns. Store undiluted at 4° C or aliquot and store at -20C for long term storage. Repeated freeze thaw cycles should be avoided.

 

 

Figure 1. Shows Neuro2A (mouse neuroblastoma) and SKNSH, 1691 and IMR32 (human neuroblastoma) cells stained with 1.11EG anti NeuroD1 monoclonal antibody by the indirect immunofluorescence method.. The secondary antibody was FITC conjugated anti-mouse Ig. Nuclear and cytoplasmic staining for NEUROD is observed in all cells. Since the cell lines tested loosely attach to surfaces of chamber slides, the cells were immobilized on adhesion slides for the staining proceedure. The cells were allowed to attach to adhesion slides, fixed in 1% paraformaldehyde, permeabilized in 0.25% Triton X 100 in PBS, blocked with 1% BSA and stained overnight with 5ug/ml antibody diluted with 1%BSA in PBS. The secondary antibody was diluted with 1% BSA in PBS according to manufacturers instruction and incubated for 2 hours.

 

 

                       

 

 

Suggested Positive control cell lines: SKNSH, Kelly, 1382.2, SHSY5Y, IMR32, 1691 neuroblastoma cells, RDES, SKES-1, SJSA Ewings sarcoma cells and SKPNDW and PFSK1 primitive neuroectodermal cells are suggested as positive control cell lines. The antibody has also reacted positively against Min 6 insulinoma cells and Neuro2A mouse cells and beta islet cells in mouse pancrease.

Figure 2 Shows K562 (chronic myelogenous leukemia), RDES (Ewings sarcoma), SKES (Ewings) sarcoma and PFSK1 (Primitive neuroectodermal tumor) cells stained with 1.11EG anti-NEUROD1 monoclonal antibody by the indirect immunoperoxidase method. K562 was used as a negative control and showed no staining whereas RDES, SKES-1 and PFSK-1 cells showed weak nuclear but strong cytoplasmic staining.

Since the cell lines tested loosely attach to surfaces of chamber slides, the cells were immobilized on adhesion slides for the staining proceedure. The cells were fixed in 1% paraformaldehyde, permeabilized in 0.25% Triton X 100 in PBS, blocked with 1% BSA and stained overnight with 5ug/ml antibody diluted with 1%BSA in PBS. The peroxidase conjugated anti mouse Ig secondary antibody was used at 1 in 1000 in 1% BSA in PBS. DAB substrate was used with nickle chloride enhancement to give dark purple/black staining for positive reaction. The substrate was added for 10 minutes, extensively washed, counterstained in diluted hematoxylene, dehydrated, cleared in xylene and mounted.

 

 

 

 

 

 

Western blotting

Figure 3. Shows reactivity of 1.11EG anti-NeuroD1 against SHSY5Y, Kelly and SKNSH neuroblastoma cell lysates by western blotting. For each cell line, a band of approximately 45kD was observed. This molecular size corresponds to the expected size for NEUROD1. The antibody was used at 2 ug/ml and incubated overnight at 4°C The peroxidase conjugated anti-mouse Ig secondary antibody was used at 1/10000 dilution and incubated for 2 hours. The blot was developed using enhanced chemiluminescence and image captured on radiographic films.

 

 

 

 

 

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Caution: The product contains 0.05% sodium azide. Sodium azide produces highly toxic hydrazoic acid under acidic conditions. It is therefore recommended to dilute azide compounds in running water before discarding to prevent the accumulation of potentially explosive deposits in the plumbing.

 

Background.

The NEUROD1 gene encodes a member of the NeuroD family of basic-loop-helix transcription factors. The NEUROD 1 was first described by Lee et al (1995) who observed that the gene was transiently expressed in subsets of neuronal cells of the central and peripheral nervous system at the time of their terminal differentiation into mature neurons. They also showed that ectopic expression of the NEUROD1 gene could convert non-neuronal cells to neuronal cells. The NEUROD1 protein forms heterodimers with other bHLH proteins and activates the transcription of a large set of genes that contain the E-box DNA sequence 5’-CANNTG’-3. NEUROD1 is involved in the regulation of several cell differentiation pathways including the development of retinal ganglion cell, inner ear sensory neurons, and endocrine islet cells. NeuroD1 regulates the expression of numerous genes including the insulin gene. Mutations in the NEUROD1 gene has been associated with type II diabetes mellitus. Defects in expression of NEUROD1 is linked with Maturity onset diabetes of the young type 6 (MODY6). The NEUROD1 protein may be localized in the cytoplasm and the nucleus. In Min 6 insulinoma cells Petersen et al (2002) were able to conclude that MEK-ERK associated phosphorylation of the serine 274 on NEUROD1 induces the translocation of the protein to the nucleus and that this may normally be associated with cell glucose concentration.

 

References

  1. Lee, J. E., Hollenberg, S. M., Snider, L., Turner, D. L., Lipnick, N., Weintraub, H. Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268: 836-844, 1995.
  2. Neurogenic differentiation 1 directs differentiation of cytokeratin 19-positive human pancreatic nonendocrine cells into insulin-producing cells. Shimoda M, et al. Transplant Proc, 2010 Jul-Aug. PMID 20692411.
  3. NeuroD1 induces terminal neuronal differentiation in olfactory neurogenesis. Boutin C, et al. Proc Natl Acad Sci USA, 2010 Jan 19. PMID2008708.
  4. Effect of upregulation of NeuroD in insulin-producing liver cells. Appavoo M, et al. Islets, 2009 Jul-Aug. PMID 21084850.
  5. Homozygous mutations in NEUROD1 are responsible for a novel syndrome of permanent neonatal diabetes and neurological abnormalities. Rubio-Cabezas O, et al. Diabetes, 2010 Sep. PMID 20573748.
  6. Combined transfection of the three transcriptional factors, PDX-1, NeuroD1, and MafA, causes differentiation of bone marrow mesenchymal stem cells into insulin-producing cells. Exp Diabetes Res. 2012;2012:672013. doi: 10.1155/2012/672013. Epub 2012 Jun Guo QS, Zhu MY, Wang L, Fan XJ, Lu YH, Wang ZW, Zhu SJ, Wang Y, Huang Y.
  7. The neuronal differentiation factor NeuroD1 downregulates the neuronal repellent factor Slit2 expression and promotes cell motility and tumor formation of neuroblastoma. Cancer Res. 2011 Apr 15;71(8):2938-48. doi: 10.1158/0008-5472.CAN-10-3524. Huang P, Kishida S, Cao D, Murakami-Tonami Y, Mu P, Nakaguro M, Koide N, Takeuchi I.
  8. Pancreatic beta cells require NeuroD to achieve and maintain functional maturity. Gu C, et al Cell Metab. 2010 Apr7. PMID20374962.
  9. Glucose induced MAPK signaling influences NeuroD1-mediated activation and nuclear localization. Helle V Petersen, Jan N Jensen, Roland Stein, Palle Serup. FEBS Letter 528 (1-3) 241-245, 2002.

 

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Title: Selective tropism of Seneca Valley virus for variant subtype small cell lung cancer.

Title: NeuroD1 regulates survival and migration of neuroendocrine lung carcinomas via signaling molecules TrkB and NCAM.

 

Title: [Expression and significance of neurogenic differentiation protein in pancreatic carcinoma].

Title: Combined transfection of the three transcriptional factors, PDX-1, NeuroD1, and MafA, causes differentiation of bone marrow mesenchymal stem cells into insulin-producing cells.

Title: Derivation of human differential photoreceptor-like cells from the iris by defined combinations of CRX, RX and NEUROD.

 

Title: Progenitor cell capacity of NeuroD1-expressing globose basal cells in the mouse olfactory epithelium.

Title: The neuronal differentiation factor NeuroD1 downregulates the neuronal repellent factor Slit2 expression and promotes cell motility and tumor formation of neuroblastoma.

 

Title: ATF2 interacts with beta-cell-enriched transcription factors, MafA, Pdx1, and beta2, and activates insulin gene transcription.

 

Title: Effect of upregulation of NeuroD in insulin-producing liver cells.

 

 


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