All lanes : Anti-NMDAR2B antibody (ab65783) at 1 µg/ml

Lane 1 : Rat Hippocampus Tissue Lysate at 10 µg
Lane 2 : Mouse Hippocampus Tissue Lysate (ab48631) at 10 µg
Lane 3 : Human brain tissue lysate - total protein (ab29466) at 20 µg
Lane 4 : Human brain hippocampus tissue lysate - total protein (ab30180) at 20 µg
Lane 5 : Human brain amygdala tissue lysate - total protein at 10 µg

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 166 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?
Additional bands at: 100 kDa, 200 kDa, 35 kDa, 45 kDa, 56 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes

This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab65783 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

All lanes : Anti-NMDAR2B antibody (ab65783) at 1 µg/ml

Lane 1 : Human brain tissue lysate - total protein (ab29466)
Lane 2 : Brain (Mouse) Tissue Lysate
Lane 3 : Brain (Rat) Tissue Lysate
Lane 4 : Hippocampus (Mouse) Tissue Lysate
Lane 5 : Rat Hippocampus Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size: 166 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?
Additional bands at: 35 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 1 minute


NMDAR2B contains a number of potential phosphorylation and glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

ab65783 staining NR2B in SKNSH cells treated with GBR 12909 dihydrochloride (ab120607), by ICC/IF. Decrease in NR2B expression correlates with increased concentration of GBR 12909 dihydrochloride, as described in literature.
The cells were incubated at 37°C for 10 minutes in media containing different concentrations of ab120607 (GBR 12909 dihydrochloride) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab65783 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

NMDAR2B was immunoprecipitated using 0.5mg Mouse Brain tissue lysate, 5µg of Rabbit polyclonal to NMDAR2B and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab65783.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 180kDa; NMDAR2B

ICC/IF image of ab65783 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab65783, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) PC12 cells at 5µg/ml.