Immunoprecipitation of Adrenergic Receptors

Phosphate-free Dulbecco's Modified Eagles Medium (DMEM) Gibco-BRL. 2. 32PiO4 radionucleotide, New England Nuclear (Boston, MA), NEX-053. 3. Lysis buffer 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride (NaF), 40 mM sodium pyrophosphate, 50 mM potassium phosphate, 10 mM sodium molybdate, 2 mM orthovanadate, 1 Triton X-100, 0.1 sodium dodecyl sulfate (SDS), leupeptin (5 J.g mL), aprotinin (5 J.g mL), benzamidine (0.1 mg mL), bacitracin (0.1 mg mL), 0.6 mM dithiothreitol...

Adrenergic Receptors

The current classification scheme for adrenergic receptors. unfortunately was prematurely identified as the a1C-AR subtype (10,11). The a1D-AR was cloned from the rat (12,13), although this receptor was prematurely called the a1A-AR. A fourth a1-AR subtype, called the a1L-(based on its low affinity for prazosin), has been suggested (14,15), although its existence is debatable (16). The evidence for a2-AR subtypes initially came from binding and functional studies in various tissues and...

Electrophoretic Transfer in a Tank System

Semi Dry Electroblotting Two Gels

During gel electrophoresis, boil the nitrocellulose, and prepare the cold transfer buffer as described in Subheading 2. 2. Transfer the proteins from gel to nitrocellulose immediately on completion of SDS-PAGE to avoid leaching of protein from the gel. 3. Place the black side cathode of the plastic sandwich transfer apparatus on the tabletop use Fig. 1 as a guide . 4. Wet the Scotch-Brite pad in transfer buffer, and place over the sandwich. Wet three pieces of 3MM filter papers, and place...

The Eukaryotic Vectors

A large variety of vectors for expression in eukaryotic cells are described in the literature. The different types of plasmids that have been used to express adrenergic receptor in eukaryotic cells can be classified into three groups, specifically monogenic, bigenic, and bicistronic vectors. The general characteristics of these plasmids are depicted in the Fig. 1. Monogenic vectors are the simplest form of expression plasmids. Briefly, they consist of a single transcription unit containing a...

Storage of Fixed Brain Sections

1. 24-Well plastic tissue-culture dishes. 2. Small glass vials with lids e.g., 22-mL scintillation vials . 3. Cryoprotectant solution 38 Combine 500 mL 0.1 M sodium phosphate buffer 500 mL dH2O, 1.59 g NaH2PO4 H2O, 5.47 g Na2HPO4 , 300 mL ethylene glycol Sigma , 300 g sucrose, 10 g polyvinylpyrolidone PVP-40 Sigma . Stir to dissolve it is difficult to get PVP into solution . Adjust volume to 1000 mL with dH2O. Store at 4 C.

Description of the Six Channel Superfusion System

Peristaltic Pump Schematic

The methods were developed using the SF-06, a six-channel superfusion system from Brandel Gaithersburg, MD . Identical methods can be used with the SF-12 or 12-channel system. The system is illustrated in Fig. 3 using a Fig. 3. Schematic of the Brandel superfusion system. For the sake of simplicity, only a single channel is depicted. Fig. 3. Schematic of the Brandel superfusion system. For the sake of simplicity, only a single channel is depicted. single channel for the sake of simplicity. A...

Detailed Description of Transcardial Perfusion for EM

Transcardial perfusion with fixatives is one of the most critical steps for successful ultrastructural preservation of tissue. The aim of transcardial perfusion is to achieve ultrastructural preservation before morphological and presumably chemical alterations of tissue are triggered by anoxia. In order to minimize artifactual alterations of tissue, anoxia may be minimized by maintaining artificial ventilation during transcardial perfusion. The other key to success is speed, i.e., minimizing...