HEK293 cells were transfected with human cDNA, and surface protein biotinylation was performed as described previously [67 (link)]. Briefly, 24 hours after transfection, cells were incubated with 1 mg/ml Sulfo-NHS-biotin for 20 min on ice, and then the biotin was quenched with 50 mM glycine. After cell lysis, protein concentrations were determined by the Bradford assay, and equal amounts of protein from each sample were added to Neutravidin beads (ThermoFisher) and rotated for 2 hr at 4°C. Total protein and pulled-down surface protein fractions were separated by SDS-PAGE, and immunoblotted using the following antibodies: mouse anti-GluN1 (BD Pharmingen, San Jose, CA, USA), rabbit anti-GluN2A (Millipore, AB1557), mouse anti-GluN2B (Alomone Labs, Jerusalem, Israel), mouse anti-transferrin receptor (Sigma), and mouse anti-tubulin. Transferrin receptor levels were used to assess whether biotin labeling was consistent across conditions. Tubulin levels were used as a loading control for total protein samples and to ensure only surface proteins were in biotinylated fractions. Chemiluminescence signals were detected with film, imaged with a Bio Rad Gel Imager, and quantified using ImageJ.
Rabbit anti glun2a
Manufactured by Merck Group
Sourced in United States
Rabbit anti-GluN2A is a laboratory reagent used for the detection and analysis of the GluN2A subunit of the NMDA receptor, which is a type of ionotropic glutamate receptor. It is a primary antibody produced in rabbits that specifically binds to the GluN2A subunit, allowing researchers to identify and study the presence and distribution of this protein in various biological samples.
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Lab products found in correlation
Mouse anti glun1, by BD (2 mentions)
Mouse anti glun2b, by BD (2 mentions)
Mouse anti gst, by Proteintech (2 mentions)
Mouse anti myc, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Rabbit anti snx27, by Proteintech (2 mentions)
Rabbit anti vps26, by Abcam (2 mentions)
Rabbit anti vps35, by Proteintech (2 mentions)
Chicken anti map2, by Abcam (2 mentions)
Rabbit anti gfp, by Proteintech (2 mentions)
Rabbit anti camkii, by Cell Signaling Technology (2 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (2 mentions)
Mouse anti gfp, by Abcam (2 mentions)
Chicken anti gfp, by Aves Labs (2 mentions)
Mouse anti glun1, by Merck Group (2 mentions)
Gel imager, by Bio-Rad (1 mentions)
Mouse anti transferrin receptor, by Merck Group (1 mentions)
Neutravidin beads, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
8 protocols using rabbit anti glun2a
1
Surface Protein Biotinylation in HEK293 Cells
2
Immunoblotting of Neuronal Proteins
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For immunoblotting, cortical neurons were harvested in sample buffer comprising 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 5% β-mercaptoethanol and heated for 5 min at 95 °C. Proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes at 80 V for 1.5 h. The membranes were incubated with 5% nonfat milk in 10 mM Tris-HCl, pH 7.4, containing 0.9% NaCl and 0.1% Tween 20 for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies. Subsequently, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5000; Pierce Biotechnology, Rockford, IL, USA). Immunoreactive proteins were detected by use of ImmunoStar basic (Wako), ImmunoStar zeta (Wako) or West Femto (Pierce Biotechnology). The following primary antibodies were used: mouse anti-β-actin (a5441, Sigma), mouse anti-GluN1 (556308, BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-GluN2A (AB1555P, Millipore), mouse anti-GluN2B (610416, BD Biosciences), and rabbit anti-calpain-2 (39165, Abcam).
3
Plasmid and Antibody Use in Neuroscience Research
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Plasmids used in this study were pGP-CMV-GCaMP6f, a gift from Douglas Kim (Addgene plasmid #40755), and pCl-SEP_GluN2B, a gift from Robert Malinow (Addgene plasmid #23998).
Primary antibodies used in this study were mouse anti-Na,K-ATPase α1 (5.7 μg/ml, DHSB), mouse anti-Na,K-ATPase α3 (1 μg/ml, Thermo Fisher Scientific), mouse anti-GluN1 (1:1000, Millipore), rabbit anti-GluN2A (2 μg/ml for immunocytochemistry, Millipore), rabbit anti-GluN2B (2 μg/ml, Millipore), mouse anti-GluN2B (2.5 μg/ml for immunocytochemistry), rabbit anti-GluN2B-pY1252/1336/1472 (1:100, Phosphosolutions), rabbit anti-GFP (4 μg/ml, Thermo Fischer Scientific), and mouse anti-Pan neuronal marker (1:1000, Millipore).
Secondary antibodies used were goat anti-mouse-Atto 488 (5 μg/ml, Sigma-Aldrich Scientific), goat anti-rabbit-Alexa 647 (10 μg/ml, Thermo Fisher Scientific), goat anti-mouse-Alexa Fluor 488 (4 μg/ml, Thermo Fisher Scientific), donkey anti-rabbit-Alexa Fluor 568 (4 μg/ml, Thermo Fisher Scientific), goat anti-rabbit-Alexa Fluor 488 (4 μg/ml, Thermo Fisher Scientific), goat anti-rabbit-Alexa Fluor 568 (4 μg/ml, Thermo Fisher Scientific), and goat anti-mouse-STAR635P (1:200, Abberior). For PLA, anti-rabbit-PLUS (Sigma Aldrich) and Anti-Mouse-MINUS (Sigma Aldrich) were used according to manufacturer’s recommendations.
Primary antibodies used in this study were mouse anti-Na,K-ATPase α1 (5.7 μg/ml, DHSB), mouse anti-Na,K-ATPase α3 (1 μg/ml, Thermo Fisher Scientific), mouse anti-GluN1 (1:1000, Millipore), rabbit anti-GluN2A (2 μg/ml for immunocytochemistry, Millipore), rabbit anti-GluN2B (2 μg/ml, Millipore), mouse anti-GluN2B (2.5 μg/ml for immunocytochemistry), rabbit anti-GluN2B-pY1252/1336/1472 (1:100, Phosphosolutions), rabbit anti-GFP (4 μg/ml, Thermo Fischer Scientific), and mouse anti-Pan neuronal marker (1:1000, Millipore).
Secondary antibodies used were goat anti-mouse-Atto 488 (5 μg/ml, Sigma-Aldrich Scientific), goat anti-rabbit-Alexa 647 (10 μg/ml, Thermo Fisher Scientific), goat anti-mouse-Alexa Fluor 488 (4 μg/ml, Thermo Fisher Scientific), donkey anti-rabbit-Alexa Fluor 568 (4 μg/ml, Thermo Fisher Scientific), goat anti-rabbit-Alexa Fluor 488 (4 μg/ml, Thermo Fisher Scientific), goat anti-rabbit-Alexa Fluor 568 (4 μg/ml, Thermo Fisher Scientific), and goat anti-mouse-STAR635P (1:200, Abberior). For PLA, anti-rabbit-PLUS (Sigma Aldrich) and Anti-Mouse-MINUS (Sigma Aldrich) were used according to manufacturer’s recommendations.
4
Antibody Validation for Neuroscience Research
5
Hippocampal Protein Expression Analysis
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Western blotting was performed as described in our previous study36 (link). Briefly, hippocampus from GH mice and 4-week SI mice were dissected, homogenized, and solubilized at 4 °C for 1 h in lysis buffer (1% CHAPS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 50 mM NaF, 1 mM Na3VO4, and protease inhibitors, pH 7.2). Bound proteins were separated by SDS PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies: goat anti-EphB1 (1:500, Santa Cruz Biotechnology, sc-68317), goat anti-EphB2 (1:1000, R&D, P54763), mouse anti-synaptophysin (1:1000, Abcam, ab8049), rabbit anti-PSD95 (1:1000; Cell Signaling Technology, 3450), mouse anti-β-actin (1:3000; Thermo Fisher Scientific, MA5-15739), rabbit anti-GluN1 (1:1000, BD, 556308), mouse anti-GluA1 (1:1000, Santa Cruz Biotechnology, sc-13152), GluA2 (1:1000, Santa Cruz Biotechnology, sc-7611), GluA6 (1:1000, Santa Cruz Biotechnology, sc-7618), rabbit anti-GluN2A (1:1000, Millipore, ab1555P), and mouse anti-GluN2B (1:1000, BD, 610417).
6
Antibody Validation for Neuroscience Research
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The rabbit polyclonal antibody against GluN2A phospho-S1459 was custom-made and has been validated previously (Mota Vieira et al., 2020 (link)). The following antibodies were purchased from commercial sources: mouse anti-β-actin (Cat# sc-47778, Santa Cruz Biotechnology), rabbit anti-CaMKII (Cat# 4436, Cell Signaling Technology), chicken anti-GFP (Cat# GFP-1020, Aves Labs); rabbit anti-GFP (Cat# 504302-AP, Proteintech), rabbit anti-GluN2A (Cat# 04-901, Millipore), mouse anti-GFP (Cat# ab1218, Abcam), mouse anti-GST (Cat# 66001-2-Ig, Proteintech), mouse anti-myc (Cat# sc-40, Santa Cruz Biotechnology), rabbit anti-SNX27 (Cat# 16329-1-AP, Proteintech), rabbit anti-VPS26 (Cat# ab181352, Abcam), rabbit anti-VPS35 (Cat# 10236-1-AP, Proteintech) and chicken anti-MAP2 (cat# ab92434, Abcam). The polyclonal rabbit antibody against GFP (JH4030) was a gift from Dr. Richard Huganir (Anggono et al., 2011 (link)). Alexa-conjugated and HRP-conjugated secondary antibodies were purchased from Thermo Scientific and GE Healthcare, respectively.
7
Western Blot Characterization of Protein Targets
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Western blot was performed as previously described (Fadó et al., 2015 (link)). Rabbit anti-CPT1C was developed in our laboratory (1:1,000; Sierra et al., 2008 (link)). Rabbit anti-ACC (1:1,000; 3676) and rabbit anti-phosphorylated ACC (Ser79; 1:2,000; 3661) were from Cell Signaling. Rabbit anti-SAC1 (1:500; 13033–1-AP) was from Proteintech. Mouse anti–β-actin (1:1,000; ab6276) and chicken anti-GFP (1:5,000; ab13970) were from Abcam. Rabbit anti-GluA1 (1:1,000; ab1504) and rabbit anti-GluA2 (1:1,000; ab397) were from Millipore. Rabbit anti-GluN2A (1:1,000; G9038), mouse anti–β-tubulin (1:2,000; T5201), and goat anti-FLAG (1:500; F3165) were from Sigma-Aldrich. HRP-conjugated secondary antibodies anti-mouse or anti-rabbit (1:10,000) were from DAKO. Blots were developed using Luminata Forte Western HRP substrate (Millipore). Semiquantitative analysis was performed using densitometry with Fiji software.
8
Immunoblot Antibody Characterization
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Rabbit anti-NPTX1, rabbit anti-NPTX2, mouse anti-NPTX2, and mouse anti-Arc were described previously (29 (link), 53 (link)). All other antibodies are from commercial companies. Sheep anti-NPTXR is from R&D Systems [catalog number: AF4414; research resource identifier (RRID): AB_2153869); mouse anti-PSD95 is from Thermo Fisher Scientific (catalog number: MA1-046; RRID: AB_2092361); mouse anti-GluN1 is from Millipore (catalog number: 05-432; RRID: AB_390129); rabbit anti-GluN2A is from Sigma-Aldrich (catalog number: G9038; RRID: AB_259980); rabbit anti-GluA4 is from Millipore (catalog number: AB1508; RRID: AB_90711); rabbit anti-HA for Western blot is from eBioscience (catalog number: 14-6756-81; RRID: AB_468301); mouse anti-HA for RiboTag pulldown is from Sigma-Aldrich (catalog number: H3663; RRID: AB_262051); mouse anti-actin is from Sigma-Aldrich (catalog number: A2228; RRID: AB_476697); ECLTM (enhanced chemiluminescence) anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) is from GE Healthcare (catalog number: NA931V); ECLTM anti-rabbit IgG HRP is from GE Healthcare (catalog number: NA934V); donkey anti-sheep IgG HRP is from Santa Cruz Biotechnology (catalog number: sc-2473; RRID: AB_641190); and mouse anti-HA is from Santa Cruz Biotechnology (catalog number: sc7392; RRID: AB_627809).
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