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Immunolocalization and Biochemical Characterization of N-methyl-D-aspartate Receptor Subunit NR1 from Rat Brain

The Protein Journal volume 25, pages 95–108 (2006)Cite this article

The N-methyl-D-aspartate (NMDA) receptor subunit NR1 gene can produce eight isoforms in rat brain. A novel methodology for purifying NMDA receptor NR1 subunit from rat brain is reported here using chicken polyclonal antibodies (IgYs) against synthetic peptides corresponding to N1, C1 and C2′ cassettes. The isolated protein was recognized by produced IgYs and commercial anti-NR1 IgGs, shown by MALDI-TOF MS a MW = 131,192 Da (glycosylated form); the enzymatically deglycosylated protein revealed a MW = 102,754 Da. The NMDA receptor NR1 subunit was characterized as being a heavily N-glycosylated protein. The isoelectric point was determined (6.3) as being different from that predicted for any of the isoforms (7.9–9.02). Attempts to separate the isoforms from the purified NR1 were unsuccessful, indicating the presence of just one isoform (NR1111). Immunohistochemistry on hippocampus regions CA1, CA3 and Dentate gyrus with anti-N1, anti-N2 and anti-C2′ IgYs showed different staining intensity, depending upon the antibody assayed.

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Abbreviations

PMSF:

phenylmethylsulfonyl fluoride

DAB:

3,3′-diaminobenzidine hydrochloride

EDTA:

ethylenediamine tetraacetic acid

SDS-PAGE:

sodium dodecyl sulphate polyacrylamide gel electrophoresis

ELISA:

enzyme linked immunoabsorbent assay

PBS:

phosphate buffer saline

ABTS:

2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium

TFA:

trifluoroacetic acid

ABC kit:

avidin–biotin complex kit

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Affiliations

  1. Biochemistry Laboratory, Chemistry Department, Universidad Nacional de Colombia, Bogotá, Colombia

    Edgar Antonio Reyes-Montaño & Gerardo Pérez-Gómez

  2. Nutrition and Biochemistry Department, Pontificia Universidad Javeriana, Bogotá, Colombia

    Leonardo René Lareo

  3. Chemistry Department, Houston University, Houston, Texas, USA

    Dar-Chone Chow

Authors
  1. Edgar Antonio Reyes-Montaño
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  2. Leonardo René Lareo
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  3. Dar-Chone Chow
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  4. Gerardo Pérez-Gómez
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Corresponding authors

Correspondence to Dar-Chone Chow or Gerardo Pérez-Gómez.

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Reyes-Montaño, E.A., Lareo, L.R., Chow, DC. et al. Immunolocalization and Biochemical Characterization of N-methyl-D-aspartate Receptor Subunit NR1 from Rat Brain. Protein J 25, 95–108 (2006). https://doi.org/10.1007/s10930-006-0001-9

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  • DOI: https://doi.org/10.1007/s10930-006-0001-9

Keywords

  • biochemical characterization
  • IgY
  • immunolocalization
  • MALDI-TOF
  • NMDA receptor
  • NR1 subunit