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Antibodies for Studying NMDA Receptor Protein Expression and,,,Synapse-Specific Immunolabeling

New antibodies for neuroscience research


Danny Q. Hoang Michael Browning Xinping Yang Peter Pingerelli
Stratagene Cloning Systems, Inc.


Stratagene introduces affinity purified antibodies for NMDA receptor subunits that are useful for studying expression of the NR2A, NR2B and NR2C subunit proteins in different cell types and brain regions. These antibodies have been used in Western blot analysis of rat brain to study expression of specific NMDA receptor subunits. In addition, these antibodies can be used for immunoprecipitation and immunohistochemistry. Stratagene also introduces an antibody for synapsin I, the synapse-specific protein, that is useful for studying synaptogenesis and synapse elimination.

Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system. The ion channels activated by glutamate are typically divided into two classes. Those that are sensitive to N-methyl-D-aspartate (NMDA) are designated NMDA receptors (NMDAR) while those activated by kainate and a-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) are known as kainate/AMPA receptors (K/AMPAR). The NMDAR plays a critical role in long-term potentiation (LTP), a putative cellular substrate of memory.1 This receptor has also been linked to conditions involving injury neurotoxicity and in pathological states, such as epilepsy and Parkinsons, Huntingtons and Alzheimers diseases.2

Molecular cloning studies indicate that five NMDA receptor genes exist, including NMDAR1 (NR1) and four NR2 subunits (NR2A-D).3 When reconstituted in vitro, the functional properties of neuronal NMDAR appear to be conferred by the combination of an NR1 subunit with any of the four NR2 subunits.4 In addition, there are a number of different splice variants of the NR1.5 Significant functional diversity has been seen in recombinant systems with heterologous coexpression of various NR2 subunits and/or NR1 splice variants.6 However, little is known about the expression of the NMDAR subunit proteins in situ.

NMDA Antibodies

Figure 1

Stratagene introduces Anti-NMDAR2A, Anti-NMDAR2B and Anti-NMDAR2C antibodies suitable for studying the expression of specific NMDAR subunit proteins in different cell types and brain regions. These antibodies were raised against fusion proteins representing selected regions of the NR2A, NR2B and NR2C subunits that have no homology with other NMDA subunits or other known protein sequences.7 The specificity of Anti-NMDAR2A and Anti-NMDAR2B was established in immunoprecipitation and Western blot experiments of whole rat brain homogenates. Figure 1 shows that Anti-NMDAR2A recognizes a single band of approximately 180 kDa in NR2A-immunoprecipitated rat brain homogenates. Similar results were obtained with Anti-NMDAR2B (figure 2). A single band of 180 kDa is observed for NR2B-immunoprecipitated rat brain homogenate blotted with Anti-NMDAR2B. As expected, no cross-reactivity was seen for the NR2B immunoprecipitation blotted with Anti-NMDAR2A. Anti-NMDAR2C recognizes the expected band (approximately 140 kDa) of NR2C as well as the 180-kDa band of NR2A and NR2B (data not shown). Thus, this antibody can serve as a pan antibody for NR2A-C.

figure 2

All three of these antibodies are suitable for Western blot, immunohistochemistry and immunoprecipitation analyses. For added convenience, Stratagene offers these antibodies with specific positive controls. Anti-NMDAR2A and Anti-NMDAR2B include a vial of rat hippocampus homogenate; Anti-NMDAR2C includes a vial of rat cerebellum homogenate.

Synapsin Antibody

figure 3

Synapsin I is a neuron-specific protein that localizes the presynaptic terminal where it associates exclusively with small synaptic vesicles. Immunolabeling of synapsin provides an ideal marker for the presynaptic terminal; previously, it has been used as a marker for synapse formation and elimination.8 Stratagene introduces an affinity-purified rabbit polyclonal synapsin antibody, Anti-Synapsin I, that can be used for studying synapsin expression in different cell types and brain regions. This antibody is suitable for studies of synapsin expression in a variety of different species, including human,9 rat,10 frog and squid.11 The specific immunoreactivity of Anti-Synapsin I is shown (figure 3). Anti-Synapsin I is useful as a synapse-specific marker that can be used to monitor synapse number and synaptogenesis. Again, Stratagene offers Anti-Synapsin I with a purified synapsin positive control, a great convenience for research applications.

Conclusions

Antibodies raised against specific peptides or fusion proteins can be used to specifically target receptor protein subunits and to localize, quantify and characterize receptor protein subunits in tissues and cell lines. Stratagene now offers sequence-specific antibodies against NR2A, NR2B and NR2C. Stratagene is also offering Anti-Synapsin I, which can be used for studying synaptogenesis and synapse elimination in a variety of species. As an added convenience, a specific positive control is included with each antibody.

Anti-NMDAR2A recognizes a predominant band, migrating at approximately 180 kDa, from a rat brain homogenate preparation. In immunoprecipitation experiments, this antibody does not cross-react with NR2B or NR2C. Strong and specific immunolabeling was also demonstrated for Anti-NMDAR2B. Anti-NMDAR2C can be used as a pan antibody for all three proteins, as it labels a 140-kDa, NR2C band and 180-kDa, NR2A and NR2B bands.

REFERENCES
  1. Collingridge, G.L., et al. (1983) J. Physiol. (Lond.) 335: 33-46.

  2. Sun, W., et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1443-1447.

  3. Moriyoshi, K., et al. (1991) Nature 354: 31-37.

  4. Monyer, H., et al. (1992) Science 256: 1217-1221.

  5. Laurie, D.J., and Seeburg, P.H. (1994) J. Neurosci. 14: 3180-3194.

  6. Monyer, H., et al. (1994) Neuron 12: 529-540.

  7. Snell, L.D., et al. (1996) Molecular Brain Research 40: 71-78.

  8. Stone, L., et al. (1994) J. Neurosci. 14: 301-309.

  9. Browning, M.D, et al. (1993) Biol. Psychol. 34: 529-535.

  10. Stone, L., et al. (1994) J. Neurosci. 14: 301-309.


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