By Min Li (Editor)
Min Li and a panel of hands-on experimentalists element cutting-edge molecular options for learning NMDA ligand-gated ion channels and constructing assays for nontherapeutic lead choice. the themes variety from cDNA cloning to in vitro and in vivo research of the channel advanced within the mammalian mind. extra subject matters contain the biochemical research of the channel protein and the development of assorted heterologous structures for either uncomplicated examine and excessive throughput monitors (HTS) for pharmaceutical chemical compounds. even supposing the focal point is on NMDA receptors, the tools are acceptable to different ligand-gated ion channels and with a few amendment might be prolonged to similar membrane signaling receptors. NMDA Receptor Protocols deals modern day scientists strong tools for simple examine on NMDA receptor constitution and serve as, in addition to huge, immense possibilities for medical research towards the advance of novel bioactive compounds.
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Extra info for NMDA Receptor Protocols (Methods in Molecular Biology Vol 128)
Example text
3. Recombinant plasmids, pJVP10Z/GluRc1, pBlueBacIII/GluR¡1, and pJVP10Z/ GluR¡2, carrying the respective inserts in the correct orientation were identified by DNA sequencing using an automatic DNA sequencer 373A (Applied Biosystems). 2. Cotransfection of Viral and Transfer Vector DNAs Using Lipofectin 1. 5 × 106 Sf9 (or Sf21) cells/well. Incubate at 27°C for 1 h to allow the cells to attach. 2. Wash the cells twice with 1 mL of serum-free TNM-FH insect medium. 3. Add 1 mL of serum-free TNM-FH insect medium.
4. 15. , St. Louis, MO, cat. no. 22-µm filter system (Corning, Boston, MA, cat. no. 430767). 16. Trypsin/EDTA solution: Add 50 mL of 10X-trypsin/EDTA (Gibco-BRL, cat. no. 5% sterile phenol red to 450 mL of PBS. Divide into 30-mL aliquots, and store frozen at –20°C for up to 6 mo. 2. Reagents and Supplies Used for Transfections 1. Twofold concentrated HEPES 42 mM HEPES-free acid (Calbiochem, La Jolla, CA, cat. no. 05. 2. One hundred-fold concentrated phosphate buffer (100X PO4): Mix 70 mM Na2HPO4 with 70 mM NaH2PO4 at 1:1 ratio.
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