重组Anti-NMDAR1抗体[EPR2481(2)] (ab109182)

Western blot: Anti-GRIN1 antibody [EPR2481(2)] (ab109182) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab109182 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, 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-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunocytochemistry/ Immunofluorescence analysis of mouse primary neuron cells labeling NMDAR1 with purified ab109182 at 1/50 (9.5µg/mL). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
Confocal scanning Z step was set as 0.3 µm followed by image processing with maximum Z projection.

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Secondary antibody - anti-rabbit HRP (ab6721)