By Stuart C. Sealfon (Eds.)

The volumes during this sequence comprise modern thoughts major to a specific department of neuroscience. they're a useful relief to the scholar in addition to the skilled researcher not just in constructing protocols in neuroscience yet in disciplines the place examine is changing into heavily with regards to neuroscience. each one quantity of Methods in Neurosciences comprises an index, and every bankruptcy contains references. Dr. Conn turned Editor-in-Chief of the sequence starting with quantity 15, so every one next quantity might be guest-edited by means of knowledgeable in that categorical box. This extra strengthens the intensity of assurance in Methods in Neurosciences for college students and researchers alike. Highlights * Cloning * Expression platforms * sign transduction * Structure-function thoughts * Antireceptor antibodies * legislation * 3D receptor modeling and computational probing

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However, a 5- to 10-fold increase in recovered [3H]spiperone binding activity was obtained when the D2 receptor was reinserted into lipid vesicles using a protocol developed earlier for/3-adrenergic receptors. Routinely, the recovery of [3H]spiperone binding activity after reconstitution was 40-50% of that originally adsorbed onto the gel. Binding of ligands to the reconstituted receptor showed the rank order and potency characteristic of binding to dopamine D2 receptors. Further modifications of the previous methodology and increasing the scale of the procedure resulted (6) in purification of D2 receptor ca.

The flow-through was collected and the gel was then rinsed with either of the above buffers at 5-10 times the applied sample volume. 0 nM [3H]bremazocine in the presence and absence of 2/xM unlabeled naloxone. The additional enrichment in binding activity after lectin chromatography was 10- to 20-fold, corresponding to an overall purification factor of 65,000-fold. 8%. The amount of affinity-purified protein from striata of five cow brains (ca. 50 g of tissue) was determined to be 60-80/xg. This material showed five bands on SDS-PAGE.

Under nonreducing conditions, a single band was seen at 50 kDa, suggesting the presence of intramolecular disulfide bonds in the native receptor. When excess free NT was added to the solubilized preparation prior to binding to the NT-Affi-Gel column, no protein bands were detected on final SDS-PAGE analysis, even on overstaining of the gel. This preblocking effect confirms that the 72-kDa polypeptide is specific to the NT receptor. The 49- and 51-kDa subunits found previously in rat brain membranes could arise from proteolytic degradation or deglycosylation of the 72-kDa protein.

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