Anti-NMDAR1 antibody [N308/48] - Neuronal Marker

• WB

Lab

Western blot - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (AB134308)

Western blot : Anti-GRIN1 antibody [N308/48] (ab134308) staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab134308 was shown to bind specifically to GRIN1. A band was observed at 120 kDa in wild-type Neuro-2a cell lysates with no signal observed at this size in GRIN1 knockout cell line ab281960 (knockout cell lysate ab282987). To generate this image, unboiled wild-type and GRIN1 knockout Neuro-2a cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (ab134308) at 1/1000 dilution

Lane 1:

Wild-type Neuro-2a cell lysate at 20 µg

Lane 2:

GRIN1 knockout Neuro-2a cell lysate at 20 µg

Lane 3:

SH-SY5Y UNBOILED cell lysate at 20 µg

Lane 4:

THP-1 UNBOILED cell lysate at 20 µg

Lane 5:

Human Brain UNBOILED cell lysate at 20 µg

Lane 6:

Human Liver UNBOILED cell lysate at 20 µg

Lane 7:

Mouse Brain UNBOILED cell lysate at 20 µg

Lane 8:

Mouse Liver UNBOILED cell lysate at 20 µg

Observed band size: 120 kDa

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• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (AB134308)

SK-N-BE cells labeling NMDAR1 using ab134308 at 1/100 dilution in ICC/IF. Cells were fixed using 4% formaldehyde for 15 minutes at room temperature. Incubated with primary antibody for 1 hour at room temperature. Secondary antibody was a goat anti-mouse ATTO 488 (green) at 1/100 dilution for 1 hour at room temperature. Counterstained with Phalloidin Texas Red F-actin stain. Nuclei were stained with DAPI (blue).

• Flow Cyt

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Flow Cytometry - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (AB134308)

Overlay histogram showing SH-SY5Y cells stained with ab134308 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab134308, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.This antibody gave a positive result in 80% methanol (5 min) fixed SH-SY5Y cells used under the same conditions. Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (AB134308)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed Neuroblastoma cells (SH-SY5Y) labeling NMDAR1 with ab134308 at 1/50 dilution. Secondary antibody : AlexaFluor® 488 at 1/1000 dilution. (A) Hoechst (blue) nuclear stain. (B) Phalloidin-iFluor 647 (red) F-Actin stain. (C) GluN1/NR1 Antibody (D) Composite.

• WB

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Western blot - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (AB134308)

All lanes:

Western blot - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (ab134308) at 1/1000 dilution

All lanes:

Rat brain membrane tissue lysate

Secondary

All lanes:

Goat anti-mouse IgG HRP

Predicted band size: 105 kDa

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• WB

CiteAb

Western blot - Anti-NMDAR1 antibody [N308/48] - Neuronal Marker (AB134308)

NMDAR1 western blot using anti-NMDAR1 antibody [N308/48] - Neuronal Marker ab134308. Publication image and figure legend from Słoniecka, M., Le Roux, S., et al., 2015, PLoS One, PubMed 26214847.

ab134308 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab134308 please see the product overview.

Glutamate and NMDAR1 expression in keratocytes.(A) Glutamate assay of cultured central and peripheral keratocytes. 250,000 cells were grown for 24h in 6 well plates in DMEM/F12 supplemented with either 2% or 0% FBS. Supernatant was collected, cell were lysed and subjected to the glutamate assay. Levels of glutamate were significantly higher in central and peripheral keratocyte culture supernatants collected from cells grown in 0% FBS than in supernatants collected from cells grown in 2% (***p<0.001 for central and ***p<0.001 for peripheral keratocytes). Levels of glutamate in cell lysates were significantly higher in peripheral keratocytes grown in 2% FBS than in peripheral keratocytes grown in 0% FBS (*p<0.05) (B) Keratocytes in sections (left panel), as well as cultured central (middle panel) and peripheral (right panel) keratocytes, were labeled with glutamate antibody (TRITC; red) and double stained with DAPI to visualize the nuclei (blue). Glutamate was expressed in both keratocytes in tissue sections and in cultured keratocytes. (C) Cornea sections (left panel), as well as cultured central (middle panel) and peripheral (right panel) keratocytes were labeled with glutamate receptor NMDAR1 (TRITC; red) and double stained with DAPI. NMDAR1 was expressed in keratocytes in tissue sections and in cultured keratocytes. (D) 250,000 central or peripheral keratocytes grown in either 2% FBS or 0% FBS were lysed and expression of NMDAR1 (105 kDa) was analyzed by western blot. NMDAR1 expression was significantly higher in both central and peripheral keratocytes grown in 2% FBS than in central and peripheral keratocytes grown in 0% FBS (*p<0.05 for central keratocytes, *p<0.05 for peripheral keratocytes). Expression of NMDAR1 was also significantly higher in central keratocytes grown in 0% FBS than in peripheral keratocytes grown in same conditions (**p<0.01). β-actin (42 kDa) served as a loading control. Values are means ± SD.

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