Glua2

Manufactured by Merck Group

Sourced in United States, Germany

GluA2 is a piece of laboratory equipment used for the detection and analysis of the GluA2 protein, which is involved in various cellular processes. The core function of GluA2 is to provide a reliable and accurate method for researchers to measure and study the expression and distribution of the GluA2 protein in various biological samples.

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Lab products found in correlation

Glua1, by Merck Group (11 mentions) Psd95, by Abcam (3 mentions) Glun2a, by Merck Group (3 mentions) Glun2b, by Merck Group (3 mentions) Glua1, by Abcam (3 mentions) Neuro 2a cells, by American Type Culture Collection (2 mentions) Anti mouse igg hrp, by Cell Signaling Technology (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Puromycin, by Merck Group (2 mentions) Glun1, by Thermo Fisher Scientific (2 mentions) Dock4, by Abcam (2 mentions) Dock4, by Thermo Fisher Scientific (2 mentions) Anti rb igg hrp, by Cell Signaling Technology (2 mentions) Nsc23766, by Selleck Chemicals (2 mentions) Mg132, by Selleck Chemicals (2 mentions) Gapdh, by Abbkine (2 mentions) Gapdh, by Merck Group (2 mentions) Glun2b, by Abcam (2 mentions) Synaptophysin, by Merck Group (2 mentions) Munc13 1, by Synaptic Systems (2 mentions)

Close spelling variants of this product

Anti-GluA2 (11 citations) GluA2/3 (8 citations) Mouse (IgG2) anti-GluA2 (4 citations) Anti-GluA2/3 (2 citations) Mouse anti-GluA2 (2 citations)

20 protocols using glua2

Western Blot Analysis of Synaptic Proteins

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The primary antibodies used in Western blot analysis were purchased from the following commercial suppliers: Dock4 (ab85723, 1:1000) and PSD95 (ab2723, 1:1000) were from Abcam; GluA1 (PC246, 1:1000), GluA2 (MAB397, 1:1000), GluN2A (AB1555P, 1:1000), GluN2B (AB1557P and 06-000, 1:1000), Rac1 (05-389, 1:2000), and puromycin (MABE343, 1:10000) were from Merck Millipore; GluN1 (32-0500, 1:1000) was from Invitrogen; Dock4 (WH0009732M1, 1:1000), ɑ-tubulin (T6199, 1:5000), and Flag (F1804, 1:1000) were from Sigma; GAPDH (A01020, 1:5000) was from Abbkine. The secondary antibodies for Western blot were purchased from Cell Signaling Technology (anti-mouse IgG-HRP, 7076s; anti-Rb IgG-HRP, 7074s). Pharmacological inhibitors MG132 and NSC23766 were purchased from Selleck. puromycin was purchased from Merck Millipore. cDNAs of Dock4 and Rac1 and their mutants, and Dock4 shRNA were described previously [27 (link), 32 (link)]. Neuro-2a cells were purchased from ATCC and were routinely tested for mycoplasma contamination-free before use.

Guo D., Peng Y., Wang L., Sun X., Wang X., Liang C., Yang X., Li S., Xu J., Ye W.C., Jiang B, & Shi L. (2019). Autism-like social deficit generated by Dock4 deficiency is rescued by restoration of Rac1 activity and NMDA receptor function. Molecular Psychiatry, 26(5), 1505-1519.

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Western Blot Analysis of Neurological Proteins

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Samples (25 μg) were loaded onto a Tris/Gly 8% gel to resolve GAPDH (37 kDa), GluA2 (100 kDa), D1 (48 kDa), and GluN2B (166 kDa), or a 4–20% gradient gel to resolve GAPDH (37 kDa), PKMζ (55 kDa)/PKCζ (70 kDa), TH (58 kDa), and DAT (50 kDa). Gels were transferred to nitrocellulose membranes and were then incubated in blocking solution containing 4% bovine serum albumin (BSA) in Tris Buffered Saline with Tween-20 (TBST; 0.1% Tween-20 in TBS) for 1 h at room temperature. Samples were incubated with the following primary antibodies overnight: GluN2B (1:1000; AbCam, Cambridge, MA), D1 (1:500; AbCam, Cambridge, MA) and with the following primary antibodies for 3 h at room temperature: PKMζ/PCKζ (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA); TH (1:2000; EMD Millipore, Billerica, MA); DAT (1:1000, Santa Cruz Biotechnology; Santa Cruz, CA); GluA2 (1:1000; EMD Millipore, Billerica, MA); and GAPDH: (1:2000, EMD Millipore; Billerica, MA). Blots were rinsed and probed with alkaline-phosphatase coupled secondary antibody and developed with BCIP/NBT substrate (KPL, Gaithersburg, MD). Membranes were scanned for quantification with NIH Image J (Rasband, 2014 ). Refer to Supplementary Figure 1 for representative immunoblots for target proteins with corresponding molecular weight markers.

Braren S.H., Drapala D., Tulloch I.K, & Serrano P.A. (2014). Methamphetamine-induced short-term increase and long-term decrease in spatial working memory affects protein Kinase M zeta (PKMζ), dopamine, and glutamate receptors. Frontiers in Behavioral Neuroscience, 8, 438.

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Comprehensive Antibody Validation for Western Blot

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The primary antibodies used in Western blot analysis were purchased from the following commercial suppliers: Dock4 (ab85723, 1:1000) and PSD95 (ab2723, 1:1000) were from Abcam; GluA1 (PC246, 1:1000), GluA2 (MAB397, 1:1000), GluN2A (AB1555P, 1:1000), GluN2B (AB1557P and 06-000, 1:1000), Rac1 (05-389, 1:2000), and puromycin (MABE343, 1:10000) were from Merck Millipore; GluN1 (32-0500, 1:1000) was from Invitrogen; Dock4 (WH0009732M1, 1:1000), ɑ-tubulin (T6199, 1:5000), and Flag (F1804, 1:1000) were from Sigma; GAPDH (A01020, 1:5000) was from Abbkine. The secondary antibodies for Western blot were purchased from Cell Signaling Technology (anti-mouse IgG-HRP, 7076s; anti-Rb IgG-HRP, 7074s). Pharmacological inhibitors MG132 and NSC23766 were purchased from Selleck. puromycin was purchased from Merck Millipore. cDNAs of Dock4 and Rac1 and their mutants, and Dock4 shRNA were described previously ^{27 (link), 32}. Neuro-2a cells were purchased from ATCC and were routinely tested for mycoplasma contamination-free before use.

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Synaptosomal Protein Analysis in Alzheimer's Mice

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Four sets of synaptosome fraction samples from 3-month-old Aβ-GFP Tg and non-Tg mice were prepared as described previously^{53 (link)}. Each set was a mixture of 3 individual Aβ-GFP Tg or non-Tg littermate hippocampi (totally 12 mice were used both in Aβ-GFP Tg or non-Tg mice). Proteins were subjected to SDS-PAGE and transferred to PVDF membranes. VAMP-2 (1:5000, Abcam, Cambridge, UK), Munc13-1(1:1000, Synaptic Systems, Gottingen, Germany), syntaxin-1 (1:1000, Merck), synaptophysin (1:2000, Sigma-Aldrich), PSD-95 (1:200, Merck), GluN1 (1:200, Merck), GluN2A (1:500, Merck), GluN2B (1:200, Merck), GluA2 (1:200, Merck), neuroligin 1(1:500, Synaptic Systems), and actin (1:2000, Merck) antibodies were used to probe the corresponding proteins and ECL was used for detection (Immunostar Zeta and Immunostar LD, FUJIFILM Wako Pure Chemical Corporation).
Immunoblotting of homogenates of individual Aβ-GFP Tg and non-Tg mouse hippocampi was performed using 6E10, GFP (1:200), GluN2B (n = 5 each for statistical analyses), tau (n = 8 each for statistical analyses, phospho T231, 1:1000, Abcam), and actin antibodies.
Immunoblot results were quantified via a C-Digit blot scanner (LI-COR Biosciences, NE, USA) and normalized with actin or GAPDH (supplementary Fig. S7) and the expression ratios of Aβ-GFP Tg were calculated with the values of non-Tg mice as 1.

Ochiishi T., Kaku M., Kiyosue K., Doi M., Urabe T., Hattori N., Shimura H, & Ebihara T. (2019). New Alzheimer’s disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid β oligomers. Scientific Reports, 9, 17368.

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Synaptic Marker Expression Analysis in Myo9a+/- Mice

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For the analysis of synaptic marker expression, hippocampi from WT and Myo9a^+/- mice were dissected and homogenized in RIPA buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40, 1% Triton X-100, protease inhibitors, pH 7.5). Ten micrograms of protein from each sample were loaded onto an acrylamide gel for western blotting_.Primary antibodies directed against the following proteins were used: Myo9a (Tü 76, WB 1:1000; Tü 78, IP 10 μg/ml; Chieregatti et al., 1998 (link); Hanley et al., 2010 (link)), Myo5a (AbCAM, 1:1000), GluA1 (Chemicon, 1:1000), GluA2 (Chemicon, 1:1000), GluA2/3 (1:2000, gift from C. Gotti), GluK2 (Prestige, 1:1000), Homer1 (Santa Cruz, 1:500), PSD95 (Neuromab, 1:20000), Synaptophysin1 (SySy, 1:5000), GAPDH (Santa Cruz, 1:1000), N-cadherin (NCAD) (AbCAM, 1:1000), PICK1 (Neuromab, 1:1000), GRIP1 (BD Transduction Laboratories, 1:2000), Tubulin (Sigma, 1:50000), Transferrin receptor (Invitrogen, 1:1000), VGAT (SySy, 1:1000), and VGLUT (SySy, 1:2000). The secondary antibodies, horseradish peroxidase- (Sigma) or IR Dyes- (LI-COR) conjugated antibodies, were used for western blots. Samples were separated using SDS-PAGE, and western blots then visualized with the Pierce ECL-Detection Kit or an Odyssey Infrared Imager (LI-COR).

Folci A., Murru L., Vezzoli E., Ponzoni L., Gerosa L., Moretto E., Longo F., Zapata J., Braida D., Pistillo F., Bähler M., Francolini M., Sala M, & Bassani S. (2016). Myosin IXa Binds AMPAR and Regulates Synaptic Structure, LTP, and Cognitive Function. Frontiers in Molecular Neuroscience, 9, 1.

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Subcellular Fractionation and Immunoblotting

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The PFC and NAc regions were dissected, homogenized in sucrose buffer containing protease inhibitors, and spun at 1000 g. The supernatant was retrieved and then spun at 10000 g. The resulting pellet was re-suspended in sucrose buffer and protease inhibitors, and spun at 10000 g. The supernatant was then discarded and the pellet containing the membrane fraction was suspended in 1% sodium dodecyl sulfate (SDS) in RIPA. Proteins were separated using 10% SDS-PAGE and transferred to PVDF membrane. The membranes were probed overnight at 4°C with primary antibodies diluted in 5% milk/Tris-buffered saline with 0.1% Tween 20. GLT-1 (1:5000, Chemicon, Billerica, MA) was measured in both the PFC and NAc, and GluA1 and GluA2 (1:1000, Chemicon, Billerica, MA) were measured in the NAc only. After incubation with HRP-conjugated secondary antiserum (Jackson Immuno; 1:10,000), immunoreactive bands on the membranes were detected by ECL Plus. Band density was measured using NIH Image J software.

LaCrosse A.L., Hill K, & Knackstedt L.A. (2015). Ceftriaxone attenuates cocaine relapse after abstinence through modulation of nucleus accumbens AMPA subunit expression. European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology, 26(2), 186-194.

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Western Blot Analysis of Neuronal Proteins

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Samples (20 μg) were loaded onto a Tris/Gly 4–20% midi gel to resolve GAPDH (37 kDa), PKMζ (55 kDa), GluA2 (102 kDa) and GR (98 kDa). Every gel contained 3–4 lanes loaded with the same control sample, all brain sample (ABS). ABS was used to standardize protein signals between gels. Gels were transferred to nitrocellulose membranes in the IBlot^® Dry Blotting System (Life Technologies; Carlsbad, CA, USA) for 9 min. Nitrocellulose membranes were then incubated in blocking solution containing 5% sucrose in Tris Buffered Saline with Tween-20 (TBST; 0.1% Tween-20 in TBS) for 30 min at room temperature. Samples were incubated with the following primary antibodies overnight: GluA2 (1:2000; Chemicon, Temecula, CA, USA), GR (1:1000, AbCam, Cambridge, MA, USA), PKMζ (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); and GAPDH: (1:2000, Chemicon, Temecula, CA, USA). Membranes were washed in TBST for 20 min and probed with Horseradish Peroxidase (HRP) conjugated secondary antibody. Membranes were incubated with Enhanced Chemiluminescence (ECL) substrate and exposed on CL-X Posure Film (Thermo Scientific; Rockford, IL, USA). Films were scanned for quantification with NIH Image J (Rasband, 2014 ).

Zanca R.M., Braren S.H., Maloney B., Schrott L.M., Luine V.N, & Serrano P.A. (2015). Environmental Enrichment Increases Glucocorticoid Receptors and Decreases GluA2 and Protein Kinase M Zeta (PKMζ) Trafficking During Chronic Stress: A Protective Mechanism?. Frontiers in Behavioral Neuroscience, 9, 303.

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Western Blot Analysis of Synaptic Proteins

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Proteins (60 μg) were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked and then incubated with primary mouse antibody to neuroligin1 (1:1000, SYSY, Göttingen, Germany) at room temperature for 2 h. Then, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:4000, ZSGB-BIO, China) for 1 h. The membrane was also blocked and incubated overnight with primary rabbit antibodies to GluA1 (1:1000, Millipore, Billerica, MA), GluA2 (1:1000, Millipore, Billerica, MA), or PSD-95 (1:500, Abcam, Cambridge, UK). Then, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000, KPL, Boston, MA). Finally, the membranes were exposed to reagents from the Enhanced Chemiluminescence Detection Kit (Thermo, Waltham, MA) and x-ray film for visualization of protein bands. The intensity of the protein bands was quantified with densitometry. The β-tubulin was used as the loading control.

Guo R., Li H., Li X., Xue Z., Sun Y., Ma D., Guan Y., Li J., Tian M, & Wang Y. (2018). Downregulation of neuroligin1 ameliorates postoperative pain through inhibiting neuroligin1/postsynaptic density 95-mediated synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor GluA1 subunits in rat dorsal horns. Molecular Pain, 14, 1744806918766745.

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Quantification of Hippocampal Synaptic Proteins

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Dorsal hippocampus of the contralateral hemisphere was microdissected and homogenized by a motor driven pestle on ice in 1× RIPA buffer (Millipore) supplemented with 1 mM PMSF, 1mM Na₃VO₂, 10 mM NaF, phosphatase inhibitor cocktail (Sigma), and Roche Complete Mini EDTA-free protease Inhibitor Cocktail Tablet (Roche). Homogenates were centrifuged 10,000 g × 10 min, and supernatants were analyzed by Western blot on Novex NuPAGE 4–12% Bis-Tris protein gels (Thermo Scientific). Membranes were washed with phosphate buffered saline with 0.05% tween-20, and blocked with 5% BSA for 1 hour at room temperature. Primary incubation was overnight at 4°C for glutamatergic receptor protein subunits GluA1 (Abcam), GluA2 (Millipore), NR2A (Millipore), NR2B (Millipore), and other synaptic proteins (Sigma) at 1:1,000 overnight, and followed by secondary antibodies (1:10,000) conjugated with IRDye 800 (mouse, LI-COR Biosciences) or IRDye 680 (rabbit, LI-COR Biosciences) for 1 hour. Protein bands were quantified by Qdyssey V3.0 software (LI-COR Biosciences).

Woodward N., Pakbin P., Saffari A., Shirmohammadi F., Haghani A., Sioutas C., Cacciottolo M., Morgan T, & Finch C. (2017). Traffic-related air pollution impact on mouse brain accelerates myelin and neuritic aging changes with specificity for CA1 neurons. Neurobiology of aging, 53, 48-58.

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Pharmacological Agents in Synaptic Regulation

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Primary antibodies used in this study include rabbit antibodies against phospho-S831 (pS831, PhosphoSolutions, Aurora, CO), phospho-S845 (pS845, PhosphoSolutions), GluA1 (Millipore), PKA catalytic subunit a (PKA Cα; Cell Signaling Technology, Danvers, MA), or PKA regulatory subunit IIβ (PKA RIIβ; Abcam, Cambridge, MA), or mouse antibodies against GluA2 (Millipore), GluA3 (Millipore), or tubulin (Millipore). Pharmacological agents, including (±)-6-chloro-PB hydrobromide (SKF81297), (-)-quinpirole hydrochloride, R(+)-SCH23390 hydrochloride, S-(-)-eticlopride hydrochloride and (-)-scopolamine hydrobromide, were purchased from Sigma-Aldrich. VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] was purchased from Axon Medchem (Reston, VA). All agents were freshly prepared at the day of experiments. VU0152100 was dissolved in 10% Tween 80 and dH₂O with the pH adjusted to approximately 7.0 using 1 N NaOH. Other agents were dissolved in physiological saline.

Xue B., Chen E.C., He N., Jin D.Z., Mao L.M, & Wang J.Q. (2016). Integrated Regulation of AMPA Glutamate Receptor Phosphorylation in the Striatum by Dopamine and Acetylcholine. Neuropharmacology, 112(Pt A), 57-65.

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