Anti-NMDAR2B antibody

4 product images

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  Immunoprecipitation - Anti-NMDAR2B antibody (ab65783)

  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) (Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) ab99697).
  Band: 180kDa; NMDAR2B

  All lanes: Immunoprecipitation - Anti-NMDAR2B antibody (ab65783)

  Predicted band size: 166 kDa

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  Western blot - Anti-NMDAR2B antibody (ab65783)

  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 ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - Anti-NMDAR2B antibody (ab65783) at 1 µg/mL

  Lane 1: Rat Hippocampus Tissue Lysate at 10 µg

  Lane 2: Mouse Hippocampus Tissue Lysate 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: 100 kDa, 180 kDa, 200 kDa, 35 kDa, 45 kDa, 56 kDa, 65 kDa

  Exposure time: 8min

• 

  Western blot - Anti-NMDAR2B antibody (ab65783)

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

  All lanes: Western blot - Anti-NMDAR2B antibody (ab65783) at 1 µg/mL

  Lane 1: Human brain tissue lysate - total protein (ab29466) at 10 µg

  Lane 2: Brain (Mouse) Tissue Lysate at 10 µg

  Lane 3: Brain (Rat) Tissue Lysate at 10 µg

  Lane 4: Hippocampus (Mouse) Tissue Lysate at 10 µg

  Lane 5: Rat Hippocampus Tissue Lysate at 10 µg

  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, 35 kDa

  Exposure time: 1min

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  Immunocytochemistry/ Immunofluorescence - Anti-NMDAR2B antibody (ab65783)

  ab65783 staining NMDAR2B in Rat Primary Neurons DIV14 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab65783 at 1µg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI (shown in blue).

  Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown..