Brain slices were stimulated with DHPG and were collected (2 slices per sample) in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). The samples were clarified by centrifugation at 14,000× g and the protein concentration was determined using a bicinchoninic acid assay kit (BCA; Thermo Scientific, Merelbeke, Belgium). The samples were heated for 10 min at 70 °C. A total of 10 μg of protein for each sample was loaded on a 7% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk, the membranes were probed overnight with anti-Arc (1/1000, Cell Signaling, Leiden, The Netherlands), anti-phospho-p44/42 MPK (pERK1/2, 1/1000, Cell Signaling Technology), anti-spectrin (1/1500, Merck Millipore, Overijse, Belgium), anti-B56α (1/250, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GluA1 (1/500, Merck Millipore), anti-GluA2 (1/1000, Merck Millipore), anti-GAPDH (1/1000, Cell Signaling Technology), or anti-β tubulin (1/1000, Neuromics, Edina, MN, USA). The membranes were then incubated with secondary antibodies coupled to peroxidase (Dako, Agilent Technologies, Diegem, Belgium) and peroxidase was detected with Pierce ECL Plus (Thermo Scientific) on ECL hyperfilm.
Anti glua1
Manufactured by Merck Group
Sourced in United States
Anti-GluA1 is a laboratory reagent used for the detection and quantification of GluA1 proteins, which are a subunit of the AMPA-type glutamate receptor. It is a specific antibody that binds to the GluA1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti glua2, by Merck Group (5 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti actin, by Abcam (3 mentions)
Anti phosphorylated glua1 s845, by Merck Group (3 mentions)
Anti glua2, by Abcam (3 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (2 mentions)
Anti β tubulin, by Neuromics (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti psd95, by Cell Signaling Technology (2 mentions)
Anti aβ 6e10, by Fortrea (2 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Anti spectrin, by Merck Group (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Ecl prime reagent, by GE Healthcare (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Perfect block, by MoBiTec (1 mentions)
Anti glun2a, by Merck Group (1 mentions)
Anti mglur5, by Cell Signaling Technology (1 mentions)
13 protocols using anti glua1
1
Western Blot Analysis of Brain Slices
2
Western Blot Analysis of Protein Targets
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Samples were separated using 4%–15% gradient gel (Biorad, USA) and proteins were transferred to PVDF membranes. Membranes were blocked with 1% blocking buffer (Perfect‐block; MoBiTec, Germany) in 0.1% TBS‐T for 1 hr and incubated overnight at 4°C with primary antibodies: anti‐c‐Myc (1:5,000 dilution; Santa Cruz Biotechnology, USA), anti‐GFP (1:5,000; Santa Cruz Biotechnology), anti‐GluA1 (1:1,000; EMD Millipore), anti‐PlexA4 (1:1,000; Abcam), anti‐Phosphorylated Ser522 of CRMP1/2 (1:1,000; Wako), and anti‐β‐actin (1:5,000; Sigma). Membranes were subsequently washed in TBS‐T and placed in HRP‐conjugated anti‐rabbit secondary antibody at a 1:1,000 dilution. After washing, membranes were reacted with ECL or ECL‐prime reagents (GE Healthcare, USA). The chemiluminescence signals on the ECL‐treated membrane were detected using LAS4000 (Fujifilm, Japan).
3
Quantifying Hippocampal Glutamate Receptor Levels
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Hippocampi were collected in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS). The samples were clarified by centrifugation at 14000 g and the protein concentration was determined using bicinchoninic acid assay kit (BCA; Thermo Scientific). Samples were heated for 10 min at 70°C. 10 μg of protein for each sample was loaded on a 7% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk, membranes were probed overnight with anti mGluR5 (1/1000, Cell Signaling), anti-GluA1 (1/500, Merck), anti-GluA2 (1/1000, Merck), anti GluN2A (1/250, Merck), anti-GluN2B (1/500, BD Biosciences) or anti-β tubulin (1/10000, Neuromics). Membranes were then incubated with secondary antibodies coupled to peroxidase (Dako) and peroxidase was detected with Pierce ECL Plus (Thermo Scientific) on ECL hyperfilm.
4
Antibody Profiling for Brain Analysis
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Anti-phospho-C/EBPβ, CST, 3084s; Anti-C/EBPβ Antibody (H-7), Santa Cruz, sc-7,962; Anti-α-Tubulin, Sigma-Aldrich, T6074; Anti-beta-actin, Abcam, ab8227; Anti-Glial Fibrillary Acidic Protein (GFAP) antibody, Sigma-Aldrich, G3893; Anti-Iba1, VWR, 019–19,741; Anti-BDNF, Abcam, ab72439; Anti-BDNF, Abcam, ab72439; Anti-NeuN, Abcam, ab177487; Anti-TrkB, R&D, MAB397; Anti-pTrkB, Santa Cruz, sc-135,645; Anti-PSD95, CST, 3450; Anti-IL-6, R&D, AF506-SP; Anti-GluA1, Merck Millipore, AB1504; Anti-GluA2, Merck Millipore, AB10529.
5
Surface Biotinylation and Western Blotting
6
Western Blot Analysis of Glutamate Receptor
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Cells were washed and scraped in cold PBS, then lysed in PBS buffer containing 1% Triton-X 100, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Roche). Protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of extract from each sample were separated by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane, then immunoblotted using primary antibodies. The primary antibodies used were: anti-GluA1 (1∶300, Millipore), anti-GluA2/3 (1∶500, Millipore), anti-pS831-GluA1 (1∶500, Epitomics, Burlingame, CA), anti-pS845-GluA1 (1∶500, Epitomics), anti-LRP1 (1∶2000, made in house) ([12] (link)), anti-PSD95 (1∶1000, Cell Signaling, Danvers, MA) and anti-β actin (1∶10, 000, Sigma-Aldrich). Image acquisition and data quantification were performed on the Odyssey Imaging system (Li-Cor, Lincoln, NE).
7
Quantitative Western Blot Analysis of Mouse Brain Proteins
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Whole and synaptosomal lysates of the mouse brains were prepared as described previously (Han et al., 2009 (link); Jin et al., 2019a (link),b (link)). The antibodies used for Western blot analysis were anti-Cofilin (1:1,000, Abcam, AB42824), anti-CYFIP1 (1:1,000, Millipore, AB6046), anti-CYFIP2 (1:3,000, Abcam, AB95969), anti-GABA-A-R-β2/3 (1:1,000, NeuroMab, 75-363), anti-GAPDH (1:3,000, Cell Signaling, #2118), anti-Gephyrin (1:500, Synaptic Systems, 147-011), anti-GluA1 (1:2,000, Millipore, 04-855), anti-GluA2 (1:1,500, Millipore, MAB397), anti-GluN1 (1:1,000, Millipore, MAB363), anti-Homer1b/c (1:1,000, Synaptic Systems, 160-002), anti-Neuroligin-3 (1:1,000, NeuroMab, 75-158), anti-PSD-95 (1:2,000, Thermo Fisher Scientific, MA1-046), and anti-WAVE1 (1:1,000, NeuroMab, 75-048). Western blot images were acquired using a ChemiDoc Touch Imaging System (Bio-Rad) and quantified using ImageJ software.
8
Forelimb Reaching Task and Synaptic Protein Analysis
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Four hours following an ICV injection of saline or 50 μM FC, mice were trained on the forelimb-reaching task. On the following day, the M1 forelimb regions from both hemispheres were dissected out from two coronal slices (750 μm). The tissue was homogenized in sucrose media (0.32 M sucrose, 10 mM HEPES, and 1 mM EDTA) using a hand held homogenizer. The homogenate was spun at 1,500 rpm for 10 min at 4°C. The supernatant was then spun at 13,300 rpm for 15 min at 4°C, to separate the synaptosomal fraction. This was resuspended in ACSF and protein concentration was estimated. Synaptosomes were lysed in RIPA buffer (50 mM Tris.HCl, 25 mM NaCl, 0.1% SDS, 0.5% Na deoxycholate, 1% Triton X 100, and 0.5 M EDTA). Proteins were separated on 4–15% Bio-Rad Mini-Protean TGX Precast SDS–PAGE gels and transferred to polyvinyl difluoride membranes. The membranes were probed with anti-GluA1 (1 : 1,500, Millipore) and anti-GAPDH (1 : 4,000, Cell Signaling). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit 1 : 2,500), and bands visualized using a Cell Biosciences FluorChem HD2 system.
9
Quantitative Western Blot Analysis of Synaptic Proteins
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Protein concentrations were quantified using the Bradford Assay (Bio-Rad Protein Assay 500-0006, Munchen, Germany), and 10 μg of whole-cell proteins or brain homogenates and 5 μg of TIF-extracted proteins were separated by 10% SDS polyacrylamide gel electrophoresis. PVDF membranes were blocked in Tris-buffered saline (5% no-fat milk powder, 0.1% Tween20) (1 h, room temperature). Primary antibodies were diluted in the same buffer (incubation overnight, 4 °C) using: anti GluN2A (1 : 2000, Gibco-Invitrogen, Paisley, Scotland, UK), anti GluN2B (1 : 2000, Gibco-Invitrogen), anti GluA1 (1 : 1000, Millipore, Billerica, MA, USA), anti GluA2 (1 : 1000, Millipore), anti PSD-95 (1 : 2000, Cayman Chemical Company, Ann Arbor, MI, USA), anti drebrin (1 : 2000, Assay Design, Ann Arbor, MI, USA), P-JNK (G-7) (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), JNK (1 : 1000, Cell Signaling Technology), Cleaved caspase-3 (1 : 1000, Cell Signaling Technology). All blots were normalized to Tubulin (1 : 5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and at least three independent experiments were performed. Blots were developed using horseradish peroxidase-conjugated secondary antibodies and the ECL chemiluminescence system. Western blots were quantified by densitometry using Quantity One software (Bio-Rad, Hercules, CA, USA).
10
Quantifying NMDA Receptor Signaling
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NMDA was purchased from Tocris Bioscience. Commercial antibodies were as follows: anti-GluA1 (04-855, Millipore), anti-GluA1 (SAB5201086, Sigma), anti phospho-GluA1 (Ser831) (36-8200, Invitrogen), anti-phospho-GluA1 (Ser845) (36-8300, Invitrogen), anti-stargazin (C8206, Sigma), anti-4.1N (276103, Synaptic Systems), anti-GST (RPN1236, Amersham), anti-HA, (901501, Covance), anti-FLAG (F7425, Sigma), and anti-MAP-2 (AB5622, Millipore) antibodies; Alexa 350 (A-11045, Thermo Fisher), Alexa 405 (A-31556, Thermo Fisher), Alexa 488 (A-11008, Thermo Fisher), Alexa 546 (A-11003, Thermo Fisher), HRP (18-8816-33, 18-8817-33, Rockland) conjugated secondary antibodies, and pre-immune IgG (CYP450-GP HU-A000).
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