Sodium dodecyl sulfate–poly acrylamide gel electrophoresis (10%) was used for Western blotting as described previously [40 (link)]. Each gel contained 4 different μg loads (2, 4, 8 and 16 μg/well) of standards, obtained from homogenate prepared by combining caudal cortices from all young control animals. Protein samples from representatives of each different age/treatment group were loaded on each gel and analyzed in triplicate. Proteins were transferred to PVDF membranes, blocked in Odyssey blocking buffer (LiCor, Lincoln, NE): Tris-buffered saline (TBS) (1:1, v/v) and incubated at 4°C in one of the following primary antibodies (diluted in blocking buffer): GluN2B (Millipore, Billerica, MA; 1:1000 dilution), GluN2A (Santa Cruz, Santa Cruz, CA; 1:250), Fyn (Santa Cruz; 1:250), PSD-95 (ThermoScientific, Waltham, MA; 1:1000), Flotillin (Santa Cruz; 1:250) or GAPDH (Calbiochem, Millipore, 1:10000). Membranes were rinsed three times with TBS-T and incubated in fluorescence-based secondary antibody (Rockland Immunochemicals, Gibertsville, PA; 1:5000) diluted in blocking buffer. Bands were visualized by scanning in the LI-COR Odyssey imager.
Glun2a
Manufactured by Santa Cruz Biotechnology
Sourced in United States
GluN2A is a subunit of the NMDA receptor, which is a type of ionotropic glutamate receptor. The NMDA receptor is a critical component of excitatory neurotransmission in the central nervous system. GluN2A plays a key role in the functional properties and regulation of the NMDA receptor.
Automatically generated - may contain errors
Lab products found in correlation
Glun2b, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Psd 95, by Thermo Fisher Scientific (1 mentions)
Glun2b, by Merck Group (1 mentions)
Glun1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Glun1, by Thermo Fisher Scientific (1 mentions)
Ephb2, by R&D Systems (1 mentions)
Ecl detection system, by Beyotime (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
Gapdh, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Cell Signaling Technology (1 mentions)
Primary antibody against total akt, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
5 protocols using glun2a
1
Western Blotting Protocol for Quantifying Protein Levels
2
Subcellular Fractionation of Pcdh10 Amygdala
3
Immunohistochemical Profiling of Rat Brain
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Rats were deeply anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brain stem was removed, post-fixed, and transferred to 25% sucrose (w/v) for cryoprotection. Transverse sections (20 -µm) were cut with a cryostat. The free-floating sections were incubated with relevant antibodies with 1% normal goat sera and 0.3% Triton x-100 overnight at 4℃. After washes in PBS, the sections were incubated with relevant IgGs conjugated to Cy3 or Cy2 (1:500; Jackson ImmunoResearch, West Grove, PA) for 4 h at room temperature or overnight at 4℃. Double immunofluorescent staining was performed for PKCγ (abcam) with NeuN (Chemicon) or GluN2A (Santa Cruz). Following washes, the stained sections were mounted on gelatin-coated slides and coverslipped with Vectashield (Vector Laboratories). Slides were examined with a Nikon fluorescence microscope and images were captured with a CCD Spot camera. Control sections were processed with the same method except that the primary antisera are omitted.
4
Western Blot Analysis of Synaptic Proteins
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After determination of the protein concentration, protein samples were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked by 5% non-fat milk in TBST, then incubated at 4 °C overnight with the following primary antibodies: EphB2 (1:1000; R&D Systems, Minneapolis, MN, USA), GluN1 (1:1000; Invitrogen), GluN2A (1:800; Santa Cruz, Dallas, TX, USA), GluN2B (1:1000; Santa Cruz), P-GluN2B (Y1472) (1:1000; Millipore, Bedford, MA, USA), P-EphB2 (Y594) (1:1000; Abcam, Cambridge, MA, USA), β-actin (1:2000; Santa Cruz), GAPDH (1:1000) and His-tag (1:1000; Zhongshan Goldenbridge Biotechnology, Beijing, China). After rinses with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000; Beyotime Institute of Biotechnology, Haimen, China). Protein bands were visualized with an ECL detection system (Beyotime Institute of Biotechnology) and quantified densitometrically with ImageJ software (NIH).
5
Western Blot Analysis of Phospho-Akt
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Western blotting assay was performed as described previously15 (link)27 (link). For the detection of phospho-Akt, the samples prepared in the same day were used. The polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA) was incubated with primary antibody against phospho-Akt (Ser473) (Cell Signaling Technology, Beverly, MA), Akt (Cell Signaling Technology, Beverly, MA), phospho-p38-MAPK (Cell Signaling Technology, Beverly, MA), p38-MAPK (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotech), GluN2A (Santa Cruz Biotech), or GluN2B (Santa Cruz Biotech). Primary antibodies were labeled with horseradish peroxidase-conjugated secondary antibody, and protein bands were imaged using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). The EC3 Imaging System (UVP, LLC, Upland, CA) was used to obtained blot images directly from the polyvinylidene difluoride membrane. For the detection of total Akt, the same polyvinylidene difluoride membrane was stripped and then re-incubated with primary antibody against total Akt (Cell Signaling Technology). The quantification of Western blot data was performed using ImageJ software.
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