New antibodies for neuroscience research
Antibodies for Studying NMDA Receptor Protein Expression and
Danny Q. Hoang • Michael Browning • Xinping Yang • Peter
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 Parkinson’s, Huntington’s and Alzheimer’s 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.
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.
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 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.
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.
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