32-0500

antibody from Invitrogen Antibodies

Targeting: GRIN1 GluN1, NMDAR1

Provider product page for 32-0500

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [38]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Immunohistochemistry [1]
Other assay [28]

Submit

Product number: 32-0500 - Provider product page
Provider: Invitrogen Antibodies
Product name: NMDAR1 Monoclonal Antibody (54.1)
Antibody type: Monoclonal
Antigen: Other
Description: This antibody specifically recognizes the ~103 kDa NMDAR1 protein. Cross-reactivity with other NMDA receptor proteins has not been observed.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 54.1
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

GRIN1 protein structure - 32-0500 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of NMDA 1 Receptor was performed by loading 30 µg of Mouse Brain (lane1) and Rat Brain (lane2) tissue lysate using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and Pierce™ Power Blotter System (22834). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk at 4ºC overnight. NMDA 1 Receptor was detected at ~103 kDa using NMDA 1 Receptor Mouse Monoclonal Antibody (Product # 32-0500) at 1-3 µg/mL in 5 % skim milk for 3 hours at room temperature on a rocking platform. Goat Anti-Mouse - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-NMDAR1 Monoclonal Antibody (54.1) (Product # 32-0500) and a 110k Da band corresponding to NMDAR1 was observed in Mouse Brain but not Mouse Liver. Tissue extracts (30 µg lysate) of Mouse Brain (Lane 1) or Mouse Liver (Lane 2) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of NMDA 1 Receptor was performed with 5 æg of the NMDA 1 Receptor Mouse Monoclonal Antibody (Product # 32-0500) on tissue extract from Rat Brain using the Dynabeads® Protein A Immunoprecipitation Kit (10006D). Normal Mouse IgG was used as a negative IP control. Subsequently, western blot analysis was performed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800). Proteins were transferred using iBlot® 2 Dry Blotting System (IB21001) to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature on a rocking platform. NMDA 1 Receptor was detected at ~ 103 kDa using NMDA 1 Receptor Mouse Monoclonal Antibody (Product # 32-0500) at 1-3 æg/mL in 5 % skim milk at 4ºC overnight on a rocking platform. Goat Anti-Mouse - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106). Lane 1 represents 10 % of the total tissue extract (input), Lane 2 is the IP performed with Mouse IgG and Lane 3 represents IP performed with NMDA 1 Receptor Mouse Monoclonal Antibody (Product # 32-0500).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 NMDA receptor subunits in spinal cord of wobbler mice . Representative photomicrographs showing NR1 (A,C) and NR2A (B,D) immunostaining in the cervical spinal cord of four-week-old wobbler mice (B). Scale bar, A, B 100 mum. C, D 20 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 western blotting analysis and quantification of the percentage of AMPA and NMDA receptor subunits expression in spinal cord of wobbler mice . Representative immunoblot from cervical spinal cord samples from 12-week-old healthy mice and age-matched wobbler mice. Left column: whole spinal cord homogenates. Right column: spinal cord TIF. Quantification of the mean values of immunodensity for CaMK, AMPA and NMDA receptor subunits, obtained from the cervical spinal wobbler mice in whole spinal cord homogenates (Homo) and in TIF. Data are representative of two independent experiments on two different homogenate preparations of four pooled animals for each group and replicated two times in each homogenate preparation. Values reported represent the percentage of the mean values of immunodensity obtained in 12-week-old wobbler mice compared to the levels measured in healthy littermates and normalized to 100. Both preparations samples were analyzed by Western blot analysis with CaMKII, GluR1, GluR2, GluR3, GluR4, NR1, and NR2A antibodies. The same amount of protein was loaded per lane.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Native supermolecular assembly of NMDAR subunits in the mammalian brain. ( a ) Schematic showing the higher-order assembly from individual proteins to complexes and supercomplexes (complexes of complexes). ( b ) Native protein complexes of GluN1, GluN2A and GluN2B detected in BNP immunoblot screen of mouse forebrain extracts. Approximately 0.8 and ~1.5 MDa complexes indicated by filled and open arrowheads, respectively (hereafter used to label all figures). The expected size of each protein in monomeric form indicated with pink rectangle. On left side, non-denaturing molecular mass indicated in mega-Daltons (MDa). ( c ) BNP GluN1 immunoblot of fractions from glycerol gradient (10-30%) ultracentrifugation. 'IN', forebrain extract supernatant. On right side, non-denaturing molecular mass indicated in MDa. ( d ) BNP GluN1 immunoblot of fresh human cortical biopsy samples from the inferior frontal (inf. front.), inferior temporal (inf. temp.) and superior frontal (sup. front.) lobes. Mouse forebrain extract supernatant shown for comparison. These data show the ~0.8 and ~1.5 MDa NMDA receptor complexes (1.5-NR and 0.8-NR) were conserved between mouse and humans.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Generation of TAP-tagged GluN1 ( Glun1 TAP/TAP ) knock-in mice for purification of native NMDARs. ( a ) Genetic engineering of TAP-tags into GluN1. Left, schematic shows tetrameric NMDAR with two GluN1 subunits (grey) engineered with tandem affinity peptide (TAP-tags, magenta) on their extracellular N-termini. GluN2 subunits (GluN2A, cyan; GluN2B, orange) shown with C-terminal cytoplasmic ESDV motifs/PDZ-ligands. TAP-tag encodes 3xFlag and His x6 . Right, schematic shows gene-targeting vector carrying TAP-tag sequence in Glun1 exon1, 5' and 3' regions of homology with genome and resultant targeted allele below. The neomycin selection cassette was subsequently deleted using Cre/loxP. Grey-filled boxes, exons; magenta, TAP cassette; brown box, neo neomycin resistance cassette; brown triangle, loxP site; dotted lines, homology arms. ( b ) Purification of native NMDARs from Glun1 TAP/TAP mouse forebrains. Left, schematic of purification steps and samples corresponding to right, immunoblots of NMDAR subunits, PSD95, actin. Dissected mouse forebrains were homogenized and fractionated. Crude membrane fraction was solubilized (membrane extract) and separated by centrifugation (supernatant and pellet). Supernatant was incubated with Flag-affinity resin capturing TAP-tagged receptors with some residual Flow-through. High yields of native receptor were released (eluate) by peptide-antigen exchange. Following elution no material remained (exchanged resin). Right, purif

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Mutant mouse screen of NMDAR supermolecular assembly. ( a ) Left, BNP GluN1 immunoblot of forebrain extracts WT (lanes 1 and 2, duplicates), Psd95 -/- (lanes 3 and 4), Psd93 -/- (lanes 5 and 6). Right panel, quantification shows relative to WT ( n =6) 1.5-NR decreased to 17% ( P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 The GluN2A/GluN2B relative molar abundance using mouse genetics and quantitative tagging. ( a ) SDS-PAGE immunoblot of WT, Glun2b 2A(CTR)/2A(CTR) and Glun2a 2B(CTR)/2B(CTR) forebrain extract. Top panel, detected the GluN2A C-terminal domain (CTD). Second panel down, GluN2B CTD. Glun2a 2B(CTR)/2B(CTR) and Glun2b 2A(CTR)/2A(CTR) provided negative controls for the specificities of the GluN2A CTD and GluN2B CTD antibodies, respectively. Third, fourth and fifth panels down, detected GluN2A NTD, GluN2B NTD subunits and GluN1, respectively. Blue and orange labels indicate chimeric Glun2a and Glun2b knock-in mutations, respectively 20 . ( b ) Dilution series of Glun2a 2B(CTR)/2B(CTR) forebrain extract into that of Glun2b 2A(CTR)/2A(CTR) indicated sensitivity of quantification. Immunoblots detected GluN2A CTD (upper panel) and GluN2B CTD (lower panel). ( c ) Quantification of GluN2A CTD and GluN2B CTD immunoblots. Measurements from Glun2b 2A(CTR)/2A(CTR) ( P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 1.5-NR and 1.5-PSD95 assemble late in postnatal development. ( a ) BNP immunoblots show WT mouse forebrain extracts at developmental time points probed with antibodies against GluN1, GluN2A, GluN2B, PSD95 and PSD93. Filled arrow, ~1.5 MDa complexes that only assembly from ~P16 onwards. Sample load was normalized by forebrain mass. Molecular weight in MDa shown on right. ( b ) Quantification of changing abundance of 0.8-NR and 1.5-NR from P12 ( n =3) to P16 ( n =3) using BNP immunoblots of GluN1. 0.8-NR increased by 25%, whereas 1.5-NR increased by 445%; t -test, P >0.01; Error bars indicate s.e.m.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 GluN1 expression in LTP-stimulated rats. (a) Representative Western blot showing GluN1 (upper) and tubulin (lower) expression in laser microdissected tissue from one LTP-stimulated animal. (b) Mean relative expression of GluN1 in the molecular layer zones from LTP-stimulated and naive hemispheres, determined as a proportion of the ipsilateral inner molecular layer. (c) Mean percentage difference in tubulin-normalised expression of GluN1 in molecular layer zones of the LTP-stimulated hemisphere compared to the naive hemisphere; * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Effect of repeated mephedrone or amphetamine administration during adolescence on MMP-9 ( A ) and NMDA subunits (GluN1, GluN2A, GluN2B) and PSD-95 ( B ) expression in the prefrontal cortex (PC) and hippocampus (HC) of rats (PND 87) that underwent the Barnes maze task. Representative blots for significant changes are presented, as well as full membranes are presented in the supplementary material (Figure S1) . Data are expressed as mean +- SEM. n = 8 rats/group. * p < 0.05 vs vehicle (0.9% NaCl). V--vehicle, M--mephedrone, A--amphetamine.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Changes in the expression of NMDA receptor subunits (GluN1, GluN2A and GluN2B) in the nucleus accumbens in rats following cocaine self-administration. All data are expressed as % of control (mean +- SEM). N = 6 rats/group. * p < 0.05 vs. yoked saline. ( a ), GluN1 protein levels [% of control]; ( b ), GluN2A protein levels [% of control]. ; ( c ), GluN2B protein levels [% of control].

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 7 Fig GABAARalpha1 and NMADR1 expression by immunofluorescence and DAPI counterstaining in the CA3 field of the hippocampus. (A) Representative immunofluorescence image of the CA3 field. (B) NE|RS group showed significantly increased expression of NMADR1 (red), whereas expression of GABAARalpha1 (green) was significantly decreased in the RS and NE|RS groups. Error bars indicate SEM. * P < .05, ** P < .005, *** P < .001. An analysis of variance with least significant difference post hoc tests for multiple comparisons was performed to examine immunofluorescence in the CA3 field of the hippocampus. GABAARalpha1 = gamma-aminobutyric acid A receptor alpha 1; NE = noise exposure; NMDAR1 = N -methyl- d -aspartate receptor 1; RS = restraint stress; SEM = standard error of mean

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 1 Abeta oligomers characterization and effects on the synapses. a A representative Coomassie-stained polyacrylamide gel and Western blot analysis of Abeta 42-1 and oAbeta 1-42 . The image shows the presence of oligomers formation for Abeta 1-42 and monomer and aggregated forms for Abeta 42-1 . b TEM revealed globular but not fibrillar structures for oAbeta 1-42 preparation, scale bar 500 nm. c Representative confocal images of GFP-transfected primary hippocampal neurons. The analysis shows that oAbeta 1-42 (500 nM, 24 h) reduces spine density (CTRL 4.58 +- 0.18; Abeta 42-1 4.15 +- 0.12; oAbeta 1-42 3.36 +- 0.18; ** p < 0.01, *** p < 0.001; one-way ANOVA, n = 34-45). Scale bar 5 mum. d MTT test shows that exposure to oAbeta 1-42 for 24 h induces cell death starting from a concentration of 1 muM (** p < 0.01, *** p < 0.001 one-way ANOVA, oAbeta 1-42 vs CTRL, n = 4). e Representative traces of mEPSCs collected in hippocampal neurons before and after oAbeta 1-42 exposure (upper panel) and before and after chemical LTD (cLTD, lower panel). At least 21 neurons before and after each treatment (oAbeta 1-42 or cLTD) have been analyzed and the related analysis of mEPSC amplitudes, here shown as cumulative probability, includes these n of excitatory events: 1550 (before oAbeta 1-42 , black line) vs 970 (after oAbeta 1-42 , red line), CTRL vs oAbeta 1-42 p = 0.0075; 2100 (before cLTD, black line) vs 1970 (after cLTD, green line), CTRL vs cLTD p < 0.0001. f Western blot analysis of s