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Western blot analysis of glutamate receptor expression on mouse and rat brain. Mouse (left panel) and rat (right panel) brain lysate were probed with Purified Mouse Anti-Glutamate Receptor (Cat. No. 556341) at 1.0 (lanes 1), 0.25 (lanes 2), and 0.125 (lanes 3) µg/test, followed by HRP Goat Anti-Mouse Ig (Cat. No. 554002) at a 1:3000 dilution. Expression of glutamate receptor is indicated by bands at 102 and 66 kDa.
BD Pharmingen™ Purified Mouse Anti-Glutamate Receptor
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
Clone 6C4 can be used for immunohistochemistry of 4% paraformaldehyde-fixed frozen tissue sections and should be titrared in the range of 2 µg/ml. Other reported applications include western blot analysis (2 µg/ml), immunohistochemistry of 4% paraformaldehyde-fixed tissue cultured cells, post-embedding immunogold electron microscopy and radioimmunoassay.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Glutamate is a major excitatory neurotransmitter in mammalian brain. Glutametergic neurotransmission is mediated by a family of glutamate receptors which can be grouped in two classes, ionotropic (GluR) and metabotropic (mGluR) receptors. The ionotropic GluRs can be divided into two subclasses, N-methyl-D-Aspartate (NMDA) and non-NMDA receptors. Five different forms of NMDA receptors (NMDAR1, R2A, R2B, R2C, and R2D) have been isolated. NMDAR1 is always required for the formation of functional NMDA receptors. Non-NMDA receptors can be divided into at least two subtypes, AMPA receptors which bind to methyl-4-isoxazole proprionic acid (AMPA) and kainate binding (KA or kainic acid) receptors. Multiple subunits appear to comprise the family of non-NMDA receptors. GluR1-4 receptors (also known as GluR-A, B, C, and D) preferentially bind to AMPA. KA receptors consist of five subunits: GluR5, 6, 7, KA-1, and KA-2. Functional receptors are formed by various combinations of these subunits and multiple forms of GluR are expressed in the same populations of neurons. GluR2 migrates at a molecular weight of ~102 kDa in SDS-PAGE.
Monoclonal antibody (mAb) 6C4 recognizes rat, macaque monkey, and dog GluR2. It does not cross-react with GluRs 1, 3, 4c, 5, 6, or 7. A recombinant trpE fusion protein containing the putative N-terminal portion (amino acids 175-430) of GluR2 was used as immunogen. The antibody was originally characterized by western blot analysis and immunohistochemical analysis of paraformaldehyde-fixed cells and paraformaldehyde-fixed frozen tissue sections. Its specificity for GluR2 was determined by radioimmunoassay and by western blot analysis. In western blots obtained from cells transiently transfected from with different GluRs, the antibody recognized GluR2 but not GluRs 1, 3, 4c, 5, 6, 7. Cross-species western blot reactivity was determined by using homogenates of fresh frozen tissue samples from rat, macaque monkey and dog cerebral cortex. In western blot analysis of tissue homogenates, clone 6C4 recognizes GluR2 as an ~102 kDa protein. Other proteins noted by western blot at 66 kDa and lower molecular weights appear to be breakdown products of GluR2. The specificity of 6C4 for GluR2 in rat brain tissue sections was verified by showing that antibody reactivity was blocked by preincubation with recombinant GluR2 protein but not with recombinant GluR1 or GluR3 protein. Immunohistochemical analysis using 6C4 shows that GluR2 is widely distributed at both the cellular and synaptic levels in rat hippocampus and neocortex, please refer to Vissavajjhala, et al. for additional staining information.
Development References (3)
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Huntley GW, Rogers SW, Moran T, et al. Selective distribution of kainate receptor subunit immunoreactivity in monkey neocortex revealed by a monoclonal antibody that recognizes glutamate receptor subunits GluR5/6/7. J Neurosci. 1993; 13(7):2965-2981. (Biology). View Reference
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Vissavajjhala P, Janssen WG, Hu Y, et al. Synaptic distribution of the AMPA-GluR2 subunit and its colocalization with calcium-binding proteins in rat cerebral cortex: an immunohistochemical study using a GluR2-specific monoclonal antibody. Exp Neurol. 1996; 142(2):296-312. (Clone-specific: Electron microscopy, Immunohistochemistry, Radioimmunoassay, Western blot). View Reference
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Watkins JC, Krogsgaard-Larsen P, Honore T. Structure-activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists. Trends Pharmacol Sci. 1990; 11(1):25-33. (Biology). View Reference
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